Universal donor cells

ABSTRACT

Genetically modified cells that are compatible with multiple subjects, e.g., universal donor cells, and methods of generating said genetic modified cells are provided herein. The universal donor cells comprise at least one genetic modification within or near at least one gene that encodes one or more MHC-I or MHC-II human leukocyte antigens or component or transcriptional regulator of the MHC-I or MHC-II complex, at least one genetic modification that increases the expression of at least one polynucleotide that encodes a tolerogenic factor, and optionally at least one genetic modification that increases or decreases the expression of at least one gene that encodes a survival factor.

RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application No.62/728,529, filed Sep. 7, 2018, the disclosure of which is herebyincorporated by reference in its entirety.

INCORPORATION BY REFERENCE OF SEQUENCE LISTING

This application contains a Sequence Listing that has been submitted inASCII format via EFS-Web and is hereby incorporated by reference in itsentirety. The ASCII copy, created on Sep. 4, 2019, is named CT109-U.S.Pat. No. 1,000,867-635287-Sequence-Listing_ST25.txt, and is 36 kilobytesin size.

FIELD OF THE INVENTION

The invention relates to the field of gene editing and, in someembodiments, to genetic modifications for the purposes of generatingcells that are compatible with multiple subjects, e.g., universal donorcells.

BACKGROUND

Various approaches have been proposed to overcome allogeneic rejectionof transplanted or engrafted cells including HLA-matching, blockingpathways that trigger T-cell activation with antibodies, use of acocktail of immune suppressive drugs, and autologous cell therapy.Another strategy to dampen graft rejection involves minimization ofallogenic differences between transplanted or engrafted cells and therecipient. The cell surface-expressed human leukocyte antigens (HLAs),molecules encoded by genes located in the human major histocompatibilitycomplex on chromosome 6, are the major mediators of immune rejection.Mismatch of a single HLA gene between the donor and subject can cause arobust immune response (Fleischhauer K. et al. “Bone marrow-allograftrejection by T lymphocytes recognizing a single amino acid difference inHLA-B44,” N Engl J Med., 1990, 323:1818-1822). HLA genes are dividedinto MHC class I (MHC-I) and MHC class II (MHC-II). MHC-I genes (HLA-A,HLA-B, and HLA-C) are expressed in almost all tissue cell types,presenting “non-self” antigen-processed peptides to CD8+ T cells,thereby promoting their activation to cytolytic CD8+T 70198359.4 cells.Transplanted or engrafted cells expressing “non-self” MHC-I moleculeswill cause a robust cellular immune response directed at these cells andultimately resulting in their demise by activated cytolytic CD8+ Tcells. MHC-I proteins are intimately associated withbeta-2-microglobulin (B2M) in the endoplasmic reticulum, which isessential for forming functional MHC-I molecules on the cell surface.

In contrast to the wide cellular expression of MHC-I genes, expressionof MHC-II genes is restricted to antigen-presenting cells such asdendritic cells, macrophages, and B cells. HLA antigen genes are themost polymorphic genes observed in the human genome (Rubinstein P., “HLAmatching for bone marrow transplantation—how much is enough?” N Engl JMed., 2001, 345:1842-1844). The generation of a “universal donor” cellthat is compatible with any HLA genotype provides an alternativestrategy that could resolve the immune rejection and associatedeconomical costs of current methodologies for immune evasion.

To generate such a line of universal donor cell(s), one previousapproach has been to functionally disrupt the expression of MHC-I andMHC-II class genes. This could be achieved through genetic disruption,e.g., of both genetic alleles encoding the MHC-I light chain, B2M. Theresulting B2M KO cell line and its derivatives would be expected toexhibit greatly reduced surface MHC-I and thus, reduced immunogenicityto allogeneic CD8+ T cells. The transcription activator-like effectornuclease (TALEN) targeting approach has been used to generateB2M-deficient hESC lines by deletion of a few nucleotides in exon 2 ofthe B2M gene (Lu, P. et al., “Generating hypoimmunogenic human embryonicstem cells by the disruption of beta 2-microglobulin,” Stem Cell Rev.2013, 9:806-813). Although the B2M-targeted hESC lines appeared to besurface HLA-I deficient, they were found to still contain mRNAs specificfor B2M and MHC-I. The B2M and MHC-I mRNAs were expressed at levelsequivalent to those of untargeted hESCs (both constitutive and IFN-ginduced). Thus, concern exists that these TALEN B2M-targeted hESC linesmight express residual cell surface MHC-I that would be sufficient tocause immune rejection, such as has been observed with B2M2/2 mousecells that also express B2M mRNA (Gross, R. and Rappuoli, R. “Pertussistoxin promoter sequences involved in modulation,” Proc Natl Acad Sci,1993, 90:3913-3917). Although the TALEN B2M targeted hESC lines were notexamined for off-target cleavage events, the occurrence of nonspecificcleavage when using TALENs remains a significant issue that would imposea major safety concern on their clinical use (Grau, J. et al.“TALENoffer: genome-wide TALEN off-target prediction,” Bioinformatics,2013, 29:2931-2932; Guilinger J. P. et al. “Broad specificity profilingof TALENs results in engineered nucleases with improved DNA-cleavagespecificity,” Nat Methods 2014, 11:429-435). Further, another reportgenerated IPS cells that escaped allogeneic recognition by knocking outa first B2M allele and knocking in a HLA-E gene at a second B2M allele,which resulted in surface expression of HLA-E dimers or trimers in theabsence of surface expression of HLA-A, HLA-B, or HLA-C (Gornalusse, G.G. et al., “HLA-E-expressing pluripotent stem cells escape allogeneicresponses and lysis by NK cells,” Nature Biotechnology, 2017, 35,765-773).

A potential limitation of some of the above strategies are that MHCclass I-negative cells are susceptible to lysis by natural killer (NK)cells as HLA molecules serve as major ligand inhibitors to naturalkiller (NK) cells. Host NK cells have been shown to eliminatetransplanted or engrafted B2M−/− donor cells, and a similar phenomenonoccurs in vitro with MEW class-I-negative human leukemic lines (Bix, M.et al., “Rejection of class I MHC-deficient haemopoietic cells byirradiated MHC-matched mice,” Nature, 1991, 349, 329-331; Zarcone, D. etal., “Human leukemia-derived cell lines and clones as models formechanistic analysis of natural killer cell-mediated cytotoxicity,”Cancer Res. 1987, 47, 2674-2682). Thus, there exists a need to improveupon previous methods to generate universal donor cells that can evadethe immune response as well as a need to generate cells that can survivepost-engraftment. As described herein, cell survival post-engraftmentmay be mediated by a host of other pathways independent of allogeneicrejection e.g., hypoxia, reactive oxygen species, nutrient deprivation,and oxidative stress. Also as described herein, genetic introduction ofsurvival factors (genes and/or proteins) may help cells to survivepost-engraftment. As described herein, a universal donor cell line maycombine properties that address both allogeneic rejection and survivalpost-engraftment.

SUMMARY

In some aspects, the present disclosure encompasses a method forgenerating a universal donor cell, the method comprising delivering to apluripotent stem cell (PSC) (a) an RNA-guided nuclease; (b) a guide RNA(gRNA) targeting a target site in a beta-2-microglobulin (B2M) genelocus; and (c) a vector comprising a nucleic acid, the nucleic acidcomprising (i) a nucleotide sequence homologous with a region locatedleft of the target site in the B2M gene locus, (ii) a nucleotidesequence encoding a tolerogenic factor, and (iii) a nucleotide sequencehomologous with a region located right of the target site in the B2Mgene locus, wherein the B2M gene locus is cleaved at the target site andthe nucleic acid is inserted into the B2M gene locus within 50 basepairs of the target site, thereby generating a universal donor cell,wherein the universal donor cell has increased immune evasion and/orcell survival compared to a PSC that does not comprise the nucleic acidinserted into the B2M gene locus.

In some embodiments, the gRNA comprises a nucleotide sequence comprisingat least one of SEQ ID NOS: 1-3. In some embodiments, (i) in the nucleicacid consists essentially of a nucleotide sequence of SEQ ID NO: 13, and(iii) in the nucleic acid consists essentially of a nucleotide sequenceof SEQ ID NO: 19.

In some embodiments, the tolerogenic factor is programmed death ligand 1(PD-L1) or human leukocyte antigen E (HLA-E). In some embodiments, thenucleotide sequence encoding the tolerogenic factor is operably linkedto an exogenous promoter. In some embodiments, the exogenous promoter isconstitutive, cell type-specific, tissue-type specific, or temporallyregulated. In some embodiments, the exogenous promoter is a CAGpromoter.

In some embodiments, the vector is a plasmid vector. In someembodiments, the plasmid vector comprises a nucleotide sequence of SEQID NO: 33 or SEQ ID NO: 34.

In some embodiments, the RNA-guided nuclease is a Cas9 nuclease. In someembodiments, the Cas9 nuclease is linked to at least one nuclearlocalization signal (NLS). In some embodiments, the CRISPR nuclease is aS. pyogenes Cas9.

In some embodiments, the PSC is a pluripotent stem cell (PSC), anembryonic stem cell (ESC), an adult stem cell (ASC), an inducedpluripotent stem cell (iPSC), or a hematopoietic stem and progenitorcell (HSPC). In some embodiments, the PSC is a human PSC.

In some aspects, the present disclosure provides a method for generatinga universal donor cell, the method comprising delivering to a PSC (a) anRNA-guided nuclease; (b) a gRNA targeting a target site in a B2M genelocus; wherein the gRNA comprises a nucleotide sequence of SEQ ID NO: 2;and (c) a vector comprising a nucleic acid, the nucleic acid comprising(i) a nucleotide sequence homologous with a region located left of thetarget site in the B2M gene locus that consists essentially of SEQ IDNO: 13, (ii) a nucleotide sequence encoding a tolerogenic factor, and(iii) a nucleotide sequence homologous with a region located right ofthe target site in the B2M gene locus that consists essentially of SEQID NO:19, wherein the B2M gene locus is cleaved at the target site andthe nucleic acid is inserted into the B2M gene locus within 50 basepairs of the target site, thereby generating the universal donor cell,wherein the universal donor cell has increased immune evasion and/orcell survival compared to a PSC that does not comprise the nucleic acidinserted into the B2M gene locus.

In some embodiments, the tolerogenic factor is programmed PD-L1 orHLA-E. In some embodiments, the nucleotide sequence encoding thetolerogenic factor is operably linked to an exogenous promoter. In someembodiments, the exogenous promoter is constitutive, cell type-specific,tissue-type specific, or temporally regulated. In some embodiments, theexogenous promoter is a CAG promoter.

In some embodiments, the vector is a plasmid vector. In someembodiments, the plasmid vector comprises a nucleotide sequence of SEQID NO: 33 or SEQ ID NO: 34.

In some embodiments, the RNA-guided nuclease is a Cas9 nuclease. In someembodiments, the Cas9 nuclease is linked to at least one nuclearlocalization signal (NLS). In some embodiments, the CRISPR nuclease is aS. pyogenes Cas9.

In some embodiments, the PSC is a pluripotent stem cell (PSC), anembryonic stem cell (ESC), an adult stem cell (ASC), an inducedpluripotent stem cell (iPSC), or a hematopoietic stem and progenitorcell (HSPC). In some embodiments, the PSC is a human PSC.

While the disclosure is susceptible to various modifications andalternative forms, specific embodiments thereof are shown by way ofexample in the drawings and will herein be described in detail. Itshould be understood, however, that the drawings and detaileddescription presented herein are not intended to limit the disclosure tothe particular embodiments disclosed, but on the contrary, the intentionis to cover all modifications, equivalents, and alternatives fallingwithin the spirit and scope of the present disclosure as defined by theappended claims.

Other features and advantages of this invention will become apparent inthe following detailed description of preferred embodiments of thisinvention, taken with reference to the accompanying drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1C provide specific gene editing strategies for immune evasion.FIG. 1A is a table that describes exemplary modifications for immuneevasion in specified cell types.

FIG. 1B provides exemplary strategies for modifying a B2M locus. FIG. 1Cprovides exemplary strategies for modifying HLA-A, HLA-B/C, and CIITAloci.

FIG. 2 depicts a portion of the B2M gene (SEQ ID NO: 6) and locations ofgRNAs (B2M-1, B2M-2, and B2M-3) for targeting exon 1. Also shown are thelocations of PCR primers (B2NIF2 and B2MR2).

FIGS. 3A-3C show results from screening of B2M gRNAs in a TC-1133 iPSCcell line. FIG. 3A is a graph showing indel (insertions+deletions)frequencies of each B2M gRNA. B2M-1 gRNA provided an indel frequency of2.5%±1.1% (n=2). B2M-2 gRNA provided an indel frequency of 87.6%±14.1%(n=2). B2M-3 gRNA provided an indel frequency of 63.9%±0.9% (n=2). FIGS.3B and 3C are graphs showing a summary of distribution of indel outcomesfor the B2M-2 (FIG. 3B) and B2M-3 (FIG. 3C) gRNAs.

FIGS. 4A-4B show results of B2M knock-outs (KO) in iPSCs using B2M-2gRNA. FIG. 4A is a graph showing a summary of distribution of indeloutcomes for the B2M-2 gRNA in iPSCs. FIG. 4B presents the cloneshomozygous (“Homo”) for B2M knock-out (KO) and the clones heterozygous(“Hets”) for B2M KO.

FIG. 5 shows an evaluation of B2M KO iPSC clones. All three B2M KOclones tested showed decreased mRNA expression of B2M relative to a wildtype, or unmodified cell.

FIGS. 6A-6D show expression of B2M and HLA-ABC in B2M KO iPSC clonesfollowing a 47-hour treatment with interferon-gamma. FIG. 6A presentsexpression in wild type cells. FIG. 6B shows expression in B2M KO cloneC4. FIG. 6C presents expression in B2M KO clone C9. FIG. 6D showsexpression in B2M KO clone C12.

FIGS. 7A-7D demonstrate the pluripotency of B2M KO iPSC clones throughevaluation of expression levels of SSEA-4 and TRA-1-60. FIG. 7A presentsexpression in wild type cells. FIG. 7B shows expression in B2M KO cloneC4. FIG. 7C presents expression in B2M KO clone C9. FIG. 7D showsexpression in B2M KO clone C12.

FIG. 8 shows TIDE analysis of B2M gRNA cutting in CyT49 cells. B2MgRNAs-1, -2, or -3 were tested.

FIGS. 9A-9B show the flow cytometry assessment of B2M expression withand without IFN-γ in WT CyT49 cells (FIG. 9A) and edited CyT49 cells(FIG. 9B).

FIG. 10 shows the plasmid map of B2M-CAGGS-PD-L1 donor vector for HDR.

FIG. 11 shows the flow cytometry analysis for pluripotency of B2MKO+PD-L1 KI CyT49 stem cells. The derived clones were >99% doublepositive for OCT4 and SOX2, two transcription factors vital forpluripotency. IgG was used as a negative control.

FIGS. 12A-12B show the flow cytometry analysis of WT CyT49 (FIG. 12A)and a B2M KO/PD-L1 KI (FIG. 12B) derived stem cell clones. WT cellsupregulate B2M expression in response to IFNγ. B2M KO/PD-L1 KI clonesfully express PD-L1 and do not express B2M with or without IFNγtreatment. NT-1=no treatment. INTG-1=50 ng/mL IFNγ 48 hour treatedcells.

FIG. 13 shows the plasmid map of B2M-CAGGS-HLA-E donor vector for HDR.

FIG. 14 shows the flow cytometry analysis for pluripotency of B2MKO/HLA-E KI CyT49 stem cells. The derived clones were >99% doublepositive for OCT4 and SOX2, two transcription factors vital forpluripotency. IgG was used as a negative control.

FIG. 15 shows the flow cytometry analysis of WT CyT49 and a B2M KO/HLA-EKI CyT49 stem cell clone. WT cells upregulate HLA-A,B,C expression inresponse to IFNγ. The B2M KO/HLA-E KI clone does not express HLA-A,B,Cwith or without IFNγ treatment. IFNγ=50 ng/mL. Cells were treated withIFNγ for 48 hours.

FIG. 16 shows the flow cytometry analysis for HLA-E expression of a B2MKO/HLA-E KI CyT49 stem cell clone. An unedited clone was used as acontrol for HLA-E expression.

FIG. 17 shows flow cytometry for FOXA2 and SOX17 at Stage 1 (DefinitiveEndoderm) cells differentiated from wild type, PD-L1 KI/B2M KO, or B2MKOhESCs.

FIG. 18 shows quantitative percentage of FOXA2 and SOX17 expression inStage 1 (Definitive Endoderm) cells differentiated from wild type, PD-L1KI/B2M KO, or B2M KO cells.

FIG. 19 shows quantitative percentage of CHGA, PDX1, and NKX6.1expression in Stage 4 (PEC) cells differentiated from wild type, B2M KO,PD-L1 KI/B2M KO (V1A), or HLA-E KI/B2M KO (V2A) cells.

FIG. 20 shows heterogeneous populations of cells at Stage 4 (PEC).

FIGS. 21A-21B show selected gene expression over differentiation timecourse in cells differentiated from wild type, PD-L1KI/B2MKO, or B2MKOcells (FIG. 21A) and cells differentiated from B2M KO/HLA-E KI (V2A)cells (FIG. 21B).

FIGS. 22A-22F show B2M and PD-L1 expression at PEC stage in cellsdifferentiated from wild type, PD-L1 KI/B2M KO, or B2M KO cells. FIG.22A shows B2M expression in wild type cells. FIG. 22B shows B2Mexpression in B2M KO cells. FIG. 22C shows B2M expression in PD-L1KI/B2M KO cells. FIG. 22D shows PD-L1 expression in wild type cells.

FIG. 22E shows PD-L1 expression in B2M KO cells. FIG. 22F shows PD-L1expression in PD-L1 KI/B2M KO cells.

FIGS. 23A-23F show MHC class I and class II expression at PEC stage incells differentiated from wild type, PD-L1 KI/B2M KO, or B2M KO cells.FIG. 23A shows MHC class I expression in wild type cells. FIG. 23B showsMHC class I expression in B2M KO cells.

FIG. 23C shows MHC class I expression in PD-L1 KI/B2M KO cells. FIG. 23Dshows MHC class II PD-L1 expression in wild type cells. FIG. 23E showsMHC class II expression in B2M KO cells. FIG. 23F shows MHC class IIexpression in PD-L1 KI/B2M KO cells.

FIGS. 24A-24D show flow cytometry analysis for T-cell activation usingthe CFSE proliferation assay. Human primary CD3+ T cells wereco-incubated with PEC derived from WT, B2M KO, or B2M KO/PD-L1 KI CyT49clones. FIG. 24A shows activation in wild type cells. FIG. 24B showsactivation in PD-L1 KI/B2M KO cells. FIG. 24C shows activation in B2M KOcells. FIG. 24D summarizes T-cell activation in the various cells.One-way ANOVA (α=0.05 with Dunnett's multiple comparisons test) with“CFSE-T alone” set as control. *, p<0.05; **, p<0.01; ***, p<0.001;****, p<0.0001. n.s.=not significant.

DETAILED DESCRIPTION I. Definitions

Deletion: As used herein, the term “deletion”, which may be usedinterchangeably with the terms “genetic deletion” or “knock-out”,generally refers to a genetic modification wherein a site or region ofgenomic DNA is removed by any molecular biology method, e.g., methodsdescribed herein, e.g., by delivering to a site of genomic DNA anendonuclease and at least one gRNA. Any number of nucleotides can bedeleted. In some embodiments, a deletion involves the removal of atleast one, at least two, at least three, at least four, at least five,at least ten, at least fifteen, at least twenty, or at least 25nucleotides. In some embodiments, a deletion involves the removal of10-50, 25-75, 50-100, 50-200, or more than 100 nucleotides. In someembodiments, a deletion involves the removal of an entire target gene,e.g., a B2M gene. In some embodiments, a deletion involves the removalof part of a target gene, e.g., all or part of a promoter and/or codingsequence of a B2M gene. In some embodiments, a deletion involves theremoval of a transcriptional regulator, e.g., a promoter region, of atarget gene. In some embodiments, a deletion involves the removal of allor part of a coding region such that the product normally expressed bythe coding region is no longer expressed, is expressed as a truncatedform, or expressed at a reduced level. In some embodiments, a deletionleads to a decrease in expression of a gene relative to an unmodifiedcell.

Endonuclease: As used herein, the term “endonuclease” generally refersto an enzyme that cleaves phosphodiester bonds within a polynucleotide.In some embodiments, an endonuclease specifically cleaves phosphodiesterbonds within a DNA polynucleotide. In some embodiments, an endonucleaseis a zinc finger nuclease (ZFN), transcription activator like effectornuclease (TALEN), homing endonuclease (HE), meganuclease, MegaTAL, or aCRISPR-associated endonuclease. In some embodiments, an endonuclease isa RNA-guided endonuclease. In certain aspects, the RNA-guidedendonuclease is a CRISPR nuclease, e.g., a Type II CRISPR Cas9endonuclease or a Type V CRISPR Cpf1 endonuclease. In some embodiments,an endonuclease is a Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cash, Cas7,Cas8, Cas9 (also known as Csn1 and Csx12), Cas100, Csy1, Csy2, Csy3,Cse1, Cse2, Csc1, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1,Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16,CsaX, Csx3, Csx1, Csx15, Csf1, Csf2, Csf3, Csf4, or Cpf1 endonuclease,or a homolog thereof, a recombination of the naturally occurringmolecule thereof, a codon-optimized version thereof, or a modifiedversion thereof, or combinations thereof. In some embodiments, anendonuclease may introduce one or more single-stranded breaks (SSBs)and/or one or more double-stranded breaks (DSBs).

Genetic modification: As used herein, the term “genetic modification”generally refers to a site of genomic DNA that has been geneticallyedited or manipulated using any molecular biological method, e.g.,methods described herein, e.g., by delivering to a site of genomic DNAan endonuclease and at least one gRNA. Example genetic modificationsinclude insertions, deletions, duplications, inversions, andtranslocations, and combinations thereof. In some embodiments, a geneticmodification is a deletion. In some embodiments, a genetic modificationis an insertion. In other embodiments, a genetic modification is aninsertion-deletion mutation (or indel), such that the reading frame ofthe target gene is shifted leading to an altered gene product or no geneproduct.

Guide RNA (gRNA): As used herein, the term “guide RNA” or “gRNA”generally refers to short ribonucleic acid that can interact with, e.g.,bind to, to an endonuclease and bind, or hybridize to a target genomicsite or region. In some embodiments, a gRNA is a single-molecule guideRNA (sgRNA). In some embodiments, a gRNA may comprise a spacer extensionregion. In some embodiments, a gRNA may comprise a tracrRNA extensionregion. In some embodiments, a gRNA is single-stranded. In someembodiments, a gRNA comprises naturally occurring nucleotides. In someembodiments, a gRNA is a chemically modified gRNA. In some embodiments,a chemically modified gRNA is a gRNA that comprises at least onenucleotide with a chemical modification, e.g., a 2′-O-methyl sugarmodification. In some embodiments, a chemically modified gRNA comprisesa modified nucleic acid backbone. In some embodiments, a chemicallymodified gRNA comprises a 2′-O-methyl-phosphorothioate residue. In someembodiments, a gRNA may be pre-complexed with a DNA endonuclease.

Insertion: As used herein, the term “insertion” which may be usedinterchangeably with the terms “genetic insertion” or “knock-in”,generally refers to a genetic modification wherein a polynucleotide isintroduced or added into a site or region of genomic DNA by anymolecular biological method, e.g., methods described herein, e.g., bydelivering to a site of genomic DNA an endonuclease and at least onegRNA. In some embodiments, an insertion may occur within or near a siteof genomic DNA that has been the site of a prior genetic modification,e.g., a deletion or insertion-deletion mutation. In some embodiments, aninsertion occurs at a site of genomic DNA that partially overlaps,completely overlaps, or is contained within a site of a prior geneticmodification, e.g., a deletion or insertion-deletion mutation. In someembodiments, an insertion occurs at a safe harbor locus. In someembodiments, an insertion involves the introduction of a polynucleotidethat encodes a protein of interest. In some embodiments, an insertioninvolves the introduction of a polynucleotide that encodes a tolerogenicfactor. In some embodiments, an insertion involves the introduction of apolynucleotide that encodes a survival factor. In some embodiments, aninsertion involves the introduction of an exogenous promoter, e.g., aconstitutive promoter, e.g., a CAG promoter. In some embodiments, aninsertion involves the introduction of a polynucleotide that encodes anoncoding gene. In general, a polynucleotide to be inserted is flankedby sequences (e.g., homology arms) having substantial sequence homologywith genomic DNA at or near the site of insertion.

Major histocompatibility complex class I (MHC-I): As used herein, theterms “Major histocompatibility complex class I” or “MHC-I” generallyrefer to a class of biomolecules that are found on the cell surface ofall nucleated cells in vertebrates, including mammals, e.g., humans; andfunction to display peptides of non-self or foreign antigens, e.g.,proteins, from within the cell (i.e. cytosolic) to cytotoxic T cells,e.g., CD8+ T cells, in order to stimulate an immune response. In someembodiments, a MHC-I biomolecule is a MHC-I gene or a MHC-I protein.Complexation of MHC-I proteins with beta-2 microglobulin (B2M) proteinis required for the cell surface expression of all MHC-I proteins. Insome embodiments, decreasing the expression of a MHC-I human leukocyteantigen (HLA) relative to an unmodified cell involves a decrease (orreduction) in the expression of a MHC-I gene. In some embodiments,decreasing the expression of a MHC-I human leukocyte antigen (HLA)relative to an unmodified cell involves a decrease (or reduction) in thecell surface expression of a MHC-I protein. In some embodiments, a MHC-Ibiomolecule is HLA-A (NCBI Gene ID No: 3105), HLA-B (NCBI Gene ID No:3106), HLA-C(NCBI Gene ID No: 3107), or B2M (NCBI Gene ID No: 567).

Major histocompatibility complex class II (MHC-II): As used herein, theterm “Major histocompatibility complex class II” or “MHC-II” generallyrefer to a class of biomolecules that are typically found on the cellsurface of antigen-presenting cells in vertebrates, including mammals,e.g., humans; and function to display peptides of non-self or foreignantigens, e.g., proteins, from outside of the cell (extracellular) tocytotoxic T cells, e.g., CD8+ T cells, in order to stimulate an immuneresponse. In some embodiments, an antigen-presenting cell is a dendriticcell, macrophage, or a B cell. In some embodiments, a MHC-II biomoleculeis a MHC-II gene or a MHC-II protein. In some embodiments, decreasingthe expression of a MHC-II human leukocyte antigen (HLA) relative to anunmodified cell involves a decrease (or reduction) in the expression ofa MHC-II gene. In some embodiments, decreasing the expression of aMHC-II human leukocyte antigen (HLA) relative to an unmodified cellinvolves a decrease (or reduction) in the cell surface expression of aMHC-II protein. In some embodiments, a MHC-II biomolecule is HLA-DPA(NCBI Gene ID No: 3113), HLA-DPB (NCBI Gene ID No: 3115), HLA-DMA (NCBIGene ID No: 3108), HLA-DMB (NCBI Gene ID No: 3109), HLA-DOA (NCBI GeneID No: 3111), HLA-DOB (NCBI Gene ID No: 3112), HLA-DQA (NCBI Gene ID No:3117), HLA-DQB (NCBI Gene ID No: 3119), HLA-DRA (NCBI Gene ID No: 3122),or HLA-DRB (NCBI Gene ID No: 3123).

Polynucleotide: As used herein, the term “polynucleotide”, which may beused interchangeably with the term “nucleic acid” generally refers to abiomolecule that comprises two or more nucleotides. In some embodiments,a polynucleotide comprises at least two, at least five at least ten, atleast twenty, at least 30, at least 40, at least 50, at least 100, atleast 200, at least 250, at least 500, or any number of nucleotides. Apolynucleotide may be a DNA or RNA molecule or a hybrid DNA/RNAmolecule. A polynucleotide may be single-stranded or double-stranded. Insome embodiments, a polynucleotide is a site or region of genomic DNA.In some embodiments, a polynucleotide is an endogenous gene that iscomprised within the genome of an unmodified cell or universal donorcell. In some embodiments, a polynucleotide is an exogenouspolynucleotide that is not integrated into genomic DNA. In someembodiments, a polynucleotide is an exogenous polynucleotide that isintegrated into genomic DNA. In some embodiments, a polynucleotide is aplasmid or an adeno-associated viral vector. In some embodiments, apolynucleotide is a circular or linear molecule.

Safe harbor locus: As used herein, the term “safe harbor locus”generally refers to any location, site, or region of genomic DNA thatmay be able to accommodate a genetic insertion into said location, site,or region without adverse effects on a cell. In some embodiments, a safeharbor locus is an intragenic or extragenic region. In some embodiments,a safe harbor locus is a region of genomic DNA that is typicallytranscriptionally silent. In some embodiments, a safe harbor locus is aAAVS1 (PPP1 R12C), ALB, Angptl3, ApoC3, ASGR2, CCR5, FIX (F9), G6PC,Gys2, HGD, Lp(a), Pcsk9, Serpinal, TF, or TTR locus. In someembodiments, a safe harbor locus is described in Sadelain, M. et al.,“Safe harbours for the integration of new DNA in the human genome,”Nature Reviews Cancer, 2012, Vol 12, pages 51-58.

Safety switch: As used herein, the term “safety switch” generally refersto a biomolecule that leads a cell to undergo apoptosis. In someembodiments, a safety switch is a protein or gene. In some embodiments,a safety switch is a suicide gene. In some embodiments, a safety switch,e.g., herpes simplex virus thymidine kinase (HSV-tk), leads a cell toundergo apoptosis by metabolizing a prodrug, e.g., ganciclovir. In someembodiments, the overexpressed presence of a safety switch on its ownleads a cell to undergo apoptosis. In some embodiments, a safety switchis a p53-based molecule, HSV-tk, or inducible caspase-9.

Subject: As used herein, the term “subject” refers to a mammal. In someembodiments, a subject is non-human primate or rodent. In someembodiments, a subject is a human. In some embodiments, a subject has,is suspected of having, or is at risk for, a disease or disorder. Insome embodiments, a subject has one or more symptoms of a disease ordisorder.

Survival factor: As used herein, the term “survival factor” generallyrefers to a protein (e.g., expressed by a polynucleotide as describedherein) that, when increased or decreased in a cell, enables the cell,e.g., a universal donor cell, to survive after transplantation orengraftment into a host subject at higher survival rates relative to anunmodified cell. In some embodiments, a survival factor is a humansurvival factor. In some embodiments, a survival factor is a member of acritical pathway involved in cell survival. In some embodiments, acritical pathway involved in cell survival has implications on hypoxia,reactive oxygen species, nutrient deprivation, and/or oxidative stress.In some embodiments, the genetic modification, e.g., deletion orinsertion, of at least one survival factor enables a universal donorcell to survive fora longer time period, e.g., at least 1.05, at least1.1, at least 1.25, at least 1.5, at least 2, at least 3, at least 4, atleast 5, at least 10, at least 20, or at least 50 times longer timeperiod, than an unmodified cell following engraftment. In someembodiments, a survival factor is ZNF143 (NCBI Gene ID No: 7702), TXNIP(NCBI Gene ID No: 10628), FOXO1 (NCBI Gene ID No: 2308), JNK (NCBI GeneID No: 5599), or MANF (NCBI Gene ID No: 7873). In some embodiments, asurvival factor is inserted into a cell, e.g., a universal donor cell.In some embodiments, a survival factor is deleted from a cell, auniversal donor cell. In some embodiments, an insertion of apolynucleotide that encodes MANF enables a cell, e.g., a universal donorcell, to survive after transplantation or engraftment into a hostsubject at higher survival rates relative to an unmodified cell. In someembodiments, a deletion or insertion-deletion mutation within or near aZNF143, TXNIP, FOXO1, or JNK gene enables a cell, e.g., a universaldonor cell, to survive after transplantation or engraftment into a hostsubject at higher survival rates relative to an unmodified cell.

Tolerogenic factor: As used herein, the term “tolerogenic factor”generally refers to a protein (e.g., expressed by a polynucleotide asdescribed herein) that, when increased or decreased in a cell, enablesthe cell, e.g., a universal donor cell, to inhibit or evade immunerejection after transplantation or engraftment into a host subject athigher rates relative to an unmodified cell. In some embodiments, atolerogenic factor is a human tolerogenic factor. In some embodiments,the genetic modification of at least one tolerogenic factor (e.g., theinsertion or deletion of at least one tolerogenic factor) enables acell, e.g., a universal donor cell. to inhibit or evade immune rejectionwith rates at least 1.05, at least 1.1, at least 1.25, at least 1.5, atleast 2, at least 3, at least 4, at least 5, at least 10, at least 20,or at least 50 times higher than an unmodified cell followingengraftment. In some embodiments, a tolerogenic factor is HLA-E (NCBIGene ID No: 3133), HLA-G (NCBI Gene ID No: 3135), CTLA-4 (NCBI Gene IDNo: 1493), CD47 (NCBI Gene ID No: 961), or PD-L1 (NCBI Gene ID No:29126). In some embodiments, a tolerogenic factor is inserted into acell, e.g., a universal donor cell. In some embodiments, a tolerogenicfactor is deleted from a cell, e.g., a universal donor cell. In someembodiments, an insertion of a polynucleotide that encodes HLA-E, HLA-G,CTLA-4, CD47, and/or PD-L1 enables a cell, e.g., a universal donor cell,to inhibit or evade immune rejection after transplantation orengraftment into a host subject.

Transcriptional regulator of MHC-I or MHC-II: As used herein, the term“transcriptional regulator of MHC-I or MHC-II” generally refers to abiomolecule that modulates, e.g., increases or decreases, the expressionof a MHC-I and/or MHC-II human leukocyte antigen. In some embodiments, abiomolecule is a polynucleotide, e.g., a gene, or a protein. In someembodiments, a transcriptional regulator of MHC-I or MHC-II willincrease or decrease the cell surface expression of at least one MHC-Ior MHC-II protein. In some embodiments, a transcriptional regulator ofMHC-I or MHC-II will increase or decrease the expression of at least oneMHC-I or MHC-II gene. In some embodiments, the transcriptional regulatoris CIITA (NCBI Gene ID No: 4261) or NLRC5 (NCBI Gene ID No: 84166). Insome embodiments, deletion or reduction of expression of CIITA or NLRC5decreases expression of at least one MHC-I or MHC-II gene.

Universal donor cell: As used herein, the term “universal donor cell”generally refers to a genetically modified cell that is less susceptibleto allogeneic rejection during a cellular transplant and/or demonstratesincreased survival after transplantation, relative to an unmodifiedcell. In some embodiments, a genetically modified cell as describedherein is a universal donor cell. In some embodiments, the universaldonor cell has increased immune evasion and/or cell survival compared toan unmodified cell. In some embodiments, the universal donor cell hasincreased cell survival compared to an unmodified cell. In someembodiments, a universal donor cell may be a stem cell. In someembodiments, a universal donor cell may be an embryonic stem cell (ESC),an adult stem cell (ASC), an induced pluripotent stem cell (iPSC), or ahematopoietic stem or progenitor cell (HSPC). In some embodiments, auniversal donor cell may be a differentiated cell. In some embodiments,a universal donor cell may be a somatic cell (e.g., immune systemcells). In some embodiments, a universal donor cell is administered to asubject. In some embodiments, a universal donor cell is administered toa subject who has, is suspected of having, or is at risk for a disease.In some embodiments, the universal donor cell is capable of beingdifferentiated into lineage-restricted progenitor cells or fullydifferentiated somatic cells. In some embodiments, thelineage-restricted progenitor cells are pancreatic endoderm progenitors,pancreatic endocrine progenitors, mesenchymal progenitor cells, muscleprogenitor cells, blast cells, or neural progenitor cells. In someembodiments, the fully differentiated somatic cells are endocrinesecretory cells such as pancreatic beta cells, epithelial cells,endodermal cells, macrophages, hepatocytes, adipocytes, kidney cells,blood cells, or immune system cells.

Unmodified cell: As used herein, the term “unmodified cell” refers to acell that has not been subjected to a genetic modification involving apolynucleotide or gene that encodes a MHC-I, MHC-I, transcriptionalregulator of MHC-I or MHC-II, survival factor, and/or tolerogenicfactor. In some embodiments, an unmodified cell may be a stem cell. Insome embodiments, an unmodified cell may be an embryonic stem cell(ESC), an adult stem cell (ASC), an induced pluripotent stem cell(iPSC), or a hematopoietic stem or progenitor cell (HSPC). In someembodiments, an unmodified cell may be a differentiated cell. In someembodiments, an unmodified cell may be selected from somatic cells(e.g., immune system cells, e.g., a T cell, e.g., a CD8+ T cell). If auniversal donor cell is compared “relative to an unmodified cell”, theuniversal donor cell and the unmodified cell are the same cell type orshare a common parent cell line, e.g., a universal donor iPSC iscompared relative to an unmodified iPSC.

Within or near a gene: As used herein, the term “within or near a gene”refers to a site or region of genomic DNA that is an intronic orextronic component of a said gene or is located proximal to a said gene.In some embodiments, a site of genomic DNA is within a gene if itcomprises at least a portion of an intron or exon of said gene. In someembodiments, a site of genomic DNA located near a gene may be at the 5′or 3′ end of said gene (e.g., the 5′ or 3′ end of the coding region ofsaid gene). In some embodiments, a site of genomic DNA located near agene may be a promoter region or repressor region that modulates theexpression of said gene. In some embodiments, a site of genomic DNAlocated near a gene may be on the same chromosome as said gene. In someembodiments, a site or region of genomic DNA is near a gene if it iswithin 50 Kb, 40 Kb, 30 Kb, 20 Kb, 10 Kb, 5 Kb, 1 Kb, or closer to the5′ or 3′ end of said gene (e.g., the 5′ or 3′ end of the coding regionof said gene).

II. Genome Editing Methods

Genome editing generally refers to the process of modifying thenucleotide sequence of a genome, preferably in a precise orpre-determined manner. In some embodiments, genome editing methods asdescribed herein, e.g., the CRISPR-endonuclease system, may be used togenetically modify a cell as described herein, e.g., to create auniversal donor cell. In some embodiments, genome editing methods asdescribed herein, e.g., the CRISPR-endonuclease system, may be used togenetically modify a cell as described herein, e.g., to introduce atleast one genetic modification within or near at least one gene thatdecreases the expression of one or more MHC-I and/or MHC-II humanleukocyte antigens or other components of the MHC-I or MHC-II complexrelative to an unmodified cell; to introduce at least one geneticmodification that increases the expression of at least onepolynucleotide that encodes a tolerogenic factor relative to anunmodified cell; and/or to introduce at least one genetic modificationthat increases or decreases the expression of at least one gene thatencodes a survival factor relative to an unmodified cell.

Examples of methods of genome editing described herein include methodsof using site-directed nucleases to cut deoxyribonucleic acid (DNA) atprecise target locations in the genome, thereby creating single-strandor double-strand DNA breaks at particular locations within the genome.Such breaks can be and regularly are repaired by natural, endogenouscellular processes, such as homology-directed repair (HDR) andnon-homologous end joining (NHEJ), as described in Cox et al.,“Therapeutic genome editing: prospects and challenges,”, NatureMedicine, 2015, 21(2), 121-31. These two main DNA repair processesconsist of a family of alternative pathways. NHEJ directly joins the DNAends resulting from a double-strand break, sometimes with the loss oraddition of nucleotide sequence, which may disrupt or enhance geneexpression. HDR utilizes a homologous sequence, or donor sequence, as atemplate for inserting a defined DNA sequence at the break point. Thehomologous sequence can be in the endogenous genome, such as a sisterchromatid. Alternatively, the donor sequence can be an exogenouspolynucleotide, such as a plasmid, a single-strand oligonucleotide, adouble-stranded oligonucleotide, a duplex oligonucleotide or a virus,that has regions (e.g., left and right homology arms) of high homologywith the nuclease-cleaved locus, but which can also contain additionalsequence or sequence changes including deletions that can beincorporated into the cleaved target locus. A third repair mechanism canbe microhomology-mediated end joining (MMEJ), also referred to as“Alternative NHEJ,” in which the genetic outcome is similar to NHEJ inthat small deletions and insertions can occur at the cleavage site. MMEJcan make use of homologous sequences of a few base pairs flanking theDNA break site to drive a more favored DNA end joining repair outcome,and recent reports have further elucidated the molecular mechanism ofthis process; see, e.g., Cho and Greenberg, Nature, 2015, 518, 174-76;Kent et al., Nature Structural and Molecular Biology, 2015, 22(3):230-7;Mateos-Gomez et al., Nature, 2015, 518, 254-57; Ceccaldi et al., Nature,2015, 528, 258-62. In some instances, it may be possible to predictlikely repair outcomes based on analysis of potential microhomologies atthe site of the DNA break.

Each of these genome editing mechanisms can be used to create desiredgenetic modifications. A step in the genome editing process can be tocreate one or two DNA breaks, the latter as double-strand breaks or astwo single-stranded breaks, in the target locus as near the site ofintended mutation. This can be achieved via the use of endonuclease s,as described and illustrated herein.

CRISPR Endonuclease System

The CRISPR-endonuclease system is a naturally occurring defensemechanism in prokaryotes that has been repurposed as a RNA-guidedDNA-targeting platform used for gene editing. CRISPR systems includeTypes I, II, III, IV, V, and VI systems. In some aspects, the CRISPRsystem is a Type II CRISPR/Cas9 system. In other aspects, the CRISPRsystem is a Type V CRISPR/Cprf system. CRISPR systems rely on a DNAendonuclease, e.g., Cas9, and two noncoding RNAs-crisprRNA (crRNA) andtrans-activating RNA (tracrRNA)—to target the cleavage of DNA.

The crRNA drives sequence recognition and specificity of theCRISPR-endonuclease complex through Watson-Crick base pairing, typicallywith a ˜20 nucleotide (nt) sequence in the target DNA. Changing thesequence of the 5′ 20 nt in the crRNA allows targeting of theCRISPR-endonuclease complex to specific loci. The CRISPR-endonucleasecomplex only binds DNA sequences that contain a sequence match to thefirst 20 nt of the single-guide RNA (sgRNA) if the target sequence isfollowed by a specific short DNA motif (with the sequence NGG) referredto as a protospacer adjacent motif (PAM).

TracrRNA hybridizes with the 3′ end of crRNA to form an RNA-duplexstructure that is bound by the endonuclease to form the catalyticallyactive CRISPR-endonuclease complex, which can then cleave the targetDNA.

Once the CRISPR-endonuclease complex is bound to DNA at a target site,two independent nuclease domains within the endonuclease each cleave oneof the DNA strands three bases upstream of the PAM site, leaving adouble-strand break (DSB) where both strands of the DNA terminate in abase pair (a blunt end).

In some embodiments, the endonuclease is a Cas9 (CRISPR associatedprotein 9). In some embodiments, the Cas9 endonuclease is fromStreptococcus pyogenes, although other Cas9 homologs may be used, e.g.,S. aureus Cas9, N. meningitidis Cas9, S. thermophilus CRISPR 1 Cas9, S.thermophilus CRISPR 3 Cas9, or T. denticola Cas9. In other instance s,the CRISPR endonuclease is Cpf1, e.g., L. bacterium ND2006 Cpf1 orAcidaminococcus sp. BV3L6 Cpf1. In some embodiments, the endonuclease isCas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cash, Cas7, Cas8, Cas9 (also knownas Csn1 and Csx12), Cas100, Csy1, Csy2, Csy3, Cse1, Cse2, Csc1, Csc2,Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6,Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csx1, Csx15,Csf1, Csf2, Csf3, Csf4, or Cpf1 endonuclease. In some embodiments,wild-type variants may be used. In some embodiments, modified versions(e.g., a homolog thereof, a recombination of the naturally occurringmolecule thereof, codon-optimized thereof, or modified versions thereof)of the preceding endonucleases may be used.

The CRISPR nuclease can be linked to at least one nuclear localizationsignal (NLS). The at least one NLS can be located at or within 50 aminoacids of the amino-terminus of the CRISPR nuclease and/or at least oneNLS can be located at or within 50 amino acids of the carboxy-terminusof the CRISPR nuclease.

Exemplary CRISPR/Cas polypeptides include the Cas9 polypeptides aspublished in Fonfara et al., “Phylogeny of Cas9 determines functionalexchangeability of dual-RNA and Cas9 among orthologous type IICRISPR-Cas systems,” Nucleic Acids Research, 2014, 42: 2577-2590. TheCRISPR/Cas gene naming system has undergone extensive rewriting sincethe Cas genes were discovered. Fonfara et al. also provides PAMsequences for the Cas9 polypeptides from various species.

Zinc Finger Nucleases

Zinc finger nucleases (ZFNs) are modular proteins comprised of anengineered zinc finger DNA binding domain linked to the catalytic domainof the type II endonuclease FokI. Because FokI functions only as adimer, a pair of ZFNs must be engineered to bind to cognate target“half-site” sequences on opposite DNA strands and with precise spacingbetween them to enable the catalytically active FokI dimer to form. Upondimerization of the FokI domain, which itself has no sequencespecificity per se, a DNA double-strand break is generated between theZFN half-sites as the initiating step in genome editing.

The DNA binding domain of each ZFN is typically comprised of 3-6 zincfingers of the abundant Cys2-His2 architecture, with each fingerprimarily recognizing a triplet of nucleotides on one strand of thetarget DNA sequence, although cross-strand interaction with a fourthnucleotide also can be important. Alteration of the amino acids of afinger in positions that make key contacts with the DNA alters thesequence specificity of a given finger. Thus, a four-finger zinc fingerprotein will selectively recognize a 12 bp target sequence, where thetarget sequence is a composite of the triplet preferences contributed byeach finger, although triplet preference can be influenced to varyingdegrees by neighboring fingers. An important aspect of ZFNs is that theycan be readily re-targeted to almost any genomic address simply bymodifying individual fingers. In most applications of ZFNs, proteins of4-6 fingers are used, recognizing 12-18 bp respectively. Hence, a pairof ZFNs will typically recognize a combined target sequence of 24-36 bp,not including the typical 5-7 bp spacer between half-sites. The bindingsites can be separated further with larger spacers, including 15-17 bp.A target sequence of this length is likely to be unique in the humangenome, assuming repetitive sequences or gene homologs are excludedduring the design process. Nevertheless, the ZFN protein-DNAinteractions are not absolute in their specificity so off-target bindingand cleavage events do occur, either as a heterodimer between the twoZFNs, or as a homodimer of one or the other of the ZFNs. The latterpossibility has been effectively eliminated by engineering thedimerization interface of the FokI domain to create “plus” and “minus”variants, also known as obligate heterodimer variants, which can onlydimerize with each other, and not with themselves. Forcing the obligateheterodimer prevents formation of the homodimer. This has greatlyenhanced specificity of ZFNs, as well as any other nuclease that adoptsthese FokI variants.

A variety of ZFN-based systems have been described in the art,modifications thereof are regularly reported, and numerous referencesdescribe rules and parameters that are used to guide the design of ZFNs;see, e.g., Segal et al., Proc Natl Acad Sci, 1999 96(6):2758-63; DreierB et al., J Mol Biol., 2000, 303(4):489-502; Liu Q et al., J Biol Chem.,2002, 277(6):3850-6; Dreier et al., J Biol Chem., 2005,280(42):35588-97; and Dreier et al., J Biol Chem. 2001,276(31):29466-78.

Transcription Activator-Like Effector Nucleases (TALENs)

TALENs represent another format of modular nucleases whereby, as withZFNs, an engineered DNA binding domain is linked to the FokI nucleasedomain, and a pair of TALENs operate in tandem to achieve targeted DNAcleavage. The major difference from ZFNs is the nature of the DNAbinding domain and the associated target DNA sequence recognitionproperties. The TALEN DNA binding domain derives from TALE proteins,which were originally described in the plant bacterial pathogenXanthomonas sp. TALEs are comprised of tandem arrays of 33-35 amino acidrepeats, with each repeat recognizing a single base pair in the targetDNA sequence that is typically up to 20 bp in length, giving a totaltarget sequence length of up to 40 bp. Nucleotide specificity of eachrepeat is determined by the repeat variable diresidue (RVD), whichincludes just two amino acids at positions 12 and 13. The bases guanine,adenine, cytosine and thymine are predominantly recognized by the fourRVDs: Asn-Asn, Asn-Ile, His-Asp and Asn-Gly, respectively. Thisconstitutes a much simpler recognition code than for zinc fingers, andthus represents an advantage over the latter for nuclease design.Nevertheless, as with ZFNs, the protein-DNA interactions of TALENs arenot absolute in their specificity, and TALENs have also benefitted fromthe use of obligate heterodimer variants of the FokI domain to reduceoff-target activity.

Additional variants of the FokI domain have been created that aredeactivated in their catalytic function. If one half of either a TALENor a ZFN pair contains an inactive FokI domain, then only single-strandDNA cleavage (nicking) will occur at the target site, rather than a DSB.The outcome is comparable to the use of CRISPR/Cas9 or CRISPR/Cpf1“nickase” mutants in which one of the Cas9 cleavage domains has beendeactivated. DNA nicks can be used to drive genome editing by HDR, butat lower efficiency than with a DSB. The main benefit is that off-targetnicks are quickly and accurately repaired, unlike the DSB, which isprone to NHEJ-mediated mis-repair.

A variety of TALEN-based systems have been described in the art, andmodifications thereof are regularly reported; see, e.g., Boch, Science,2009 326(5959):1509-12; Mak et al., Science, 2012, 335(6069):716-9; andMoscou et al., Science, 2009, 326(5959):1501. The use of TALENs based onthe “Golden Gate” platform, or cloning scheme, has been described bymultiple groups; see, e.g., Cermak et al., Nucleic Acids Res., 2011,39(12):e82; Li et al., Nucleic Acids Res., 2011, 39(14):6315-25; Weberet al., PLoS One., 2011, 6(2):e16765; Wang et al., J Genet Genomics,2014, 41(6):339-47.; and Cermak T et al., Methods Mol Biol., 20151239:133-59.

Homing Endonucleases

Homing endonucleases (HEs) are sequence-specific endonucleases that havelong recognition sequences (14-44 base pairs) and cleave DNA with highspecificity—often at sites unique in the genome. There are at least sixknown families of HEs as classified by their structure, includingGIY-YIG, His-Cis box, H—N—H, PD-(D/E)xK, and Vsr-like that are derivedfrom a broad range of hosts, including eukarya, protists, bacteria,archaea, cyanobacteria and phage. As with ZFNs and TALENs, HEs can beused to create a DSB at a target locus as the initial step in genomeediting. In addition, some natural and engineered HEs cut only a singlestrand of DNA, thereby functioning as site-specific nickases. The largetarget sequence of HEs and the specificity that they offer have madethem attractive candidates to create site-specific DSBs.

A variety of HE-based systems have been described in the art, andmodifications thereof are regularly reported; see, e.g., the reviews bySteentoft et al., Glycobiology, 2014, 24(8):663-80; Belfort andBonocora, Methods Mol Biol., 2014, 1123:1-26; and Hafez and Hausner,Genome, 2012, 55(8):553-69.

MegaTAL/Tev-mTALEN/MegaTev

As further examples of hybrid nucleases, the MegaTAL platform andTev-mTALEN platform use a fusion of TALE DNA binding domains andcatalytically active HEs, taking advantage of both the tunable DNAbinding and specificity of the TALE, as well as the cleavage sequencespecificity of the HE; see, e.g., Boissel et al., Nucleic Acids Res.,2014, 42: 2591-2601; Kleinstiver et al., G3, 2014, 4:1155-65; andBoissel and Scharenberg, Methods Mol. Biol., 2015, 1239: 171-96.

In a further variation, the MegaTev architecture is the fusion of ameganuclease (Mega) with the nuclease domain derived from the GIY-YIGhoming endonuclease I-Teel (Tev). The two active sites are positioned˜30 bp apart on a DNA substrate and generate two DSBs withnon-compatible cohesive ends; see, e.g., Wolfs et al., Nucleic AcidsRes., 2014, 42, 8816-29. It is anticipated that other combinations ofexisting nuclease-based approaches will evolve and be useful inachieving the targeted genome modifications described herein.

dCas9-FokI or dCpf1-FokI and Other Nucleases

Combining the structural and functional properties of the nucleaseplatforms described above offers a further approach to genome editingthat can potentially overcome some of the inherent deficiencies. As anexample, the CRISPR genome editing system typically uses a single Cas9endonuclease to create a DSB. The specificity of targeting is driven bya 20 or 24 nucleotide sequence in the guide RNA that undergoesWatson-Crick base-pairing with the target DNA (plus an additional 2bases in the adjacent NAG or NGG PAM sequence in the case of Cas9 fromS. pyogenes). Such a sequence is long enough to be unique in the humangenome, however, the specificity of the RNA/DNA interaction is notabsolute, with significant promiscuity sometimes tolerated, particularlyin the 5′ half of the target sequence, effectively reducing the numberof bases that drive specificity. One solution to this has been tocompletely deactivate the Cas9 or Cpf1 catalytic function—retaining onlythe RNA-guided DNA binding function—and instead fusing a FokI domain tothe deactivated Cas9; see, e.g., Tsai et al., Nature Biotech, 2014, 32:569-76; and Guilinger et al., Nature Biotech., 2014, 32: 577-82. BecauseFokI must dimerize to become catalytically active, two guide RNAs arerequired to tether two FokI fusions in close proximity to form the dimerand cleave DNA. This essentially doubles the number of bases in thecombined target sites, thereby increasing the stringency of targeting byCRISPR-based systems.

As further example, fusion of the TALE DNA binding domain to acatalytically active HE, such as I-TevI, takes advantage of both thetunable DNA binding and specificity of the TALE, as well as the cleavagesequence specificity of I-TevI, with the expectation that off-targetcleavage can be further reduced.

RNA-Guided Endonucleases

The RNA-guided endonuclease systems as used herein can comprise an aminoacid sequence having at least 10%, at least 15%, at least 20%, at least30%, at least 40%, at least 50%, at least 60%, at least 70%, at least75%, at least 80%, at least 85%, at least 90%, at least 95%, at least99%, or 100% amino acid sequence identity to a wild-type exemplaryendonuclease, e.g., Cas9 from S. pyogenes, US2014/0068797 Sequence IDNo. 8 or Sapranauskas et al., Nucleic Acids Res, 39(21): 9275-9282(2011). The endonuclease can comprise at least 70, 75, 80, 85, 90, 95,97, 99, or 100% identity to a wild-type endonuclease (e.g., Cas9 from S.pyogenes, supra) over 10 contiguous amino acids. The endonuclease cancomprise at most: 70, 75, 80, 85, 90, 95, 97, 99, or 100% identity to awild-type endonuclease (e.g., Cas9 from S. pyogenes, supra) over 10contiguous amino acids. The endonuclease can comprise at least: 70, 75,80, 85, 90, 95, 97, 99, or 100% identity to a wild-type endonuclease(e.g., Cas9 from S. pyogenes, supra) over 10 contiguous amino acids in aHNH nuclease domain of the endonuclease. The endonuclease can compriseat most: 70, 75, 80, 85, 90, 95, 97, 99, or 100% identity to a wild-typeendonuclease (e.g., Cas9 from S. pyogenes, supra) over 10 contiguousamino acids in a HNH nuclease domain of the endonuclease. Theendonuclease can comprise at least: 70, 75, 80, 85, 90, 95, 97, 99, or100% identity to a wild-type endonuclease (e.g., Cas9 from S. pyogenes,supra) over 10 contiguous amino acids in a RuvC nuclease domain of theendonuclease. The endonuclease can comprise at most: 70, 75, 80, 85, 90,95, 97, 99, or 100% identity to a wild-type endonuclease (e.g., Cas9from S. pyogenes, supra) over 10 contiguous amino acids in a RuvCnuclease domain of the endonuclease.

The endonuclease can comprise a modified form of a wild-type exemplaryendonuclease. The modified form of the wild-type exemplary endonucleasecan comprise a mutation that reduces the nucleic acid-cleaving activityof the endonuclease. The modified form of the wild-type exemplaryendonuclease can have less than 90%, less than 80%, less than 70%, lessthan 60%, less than 50%, less than 40%, less than 30%, less than 20%,less than 10%, less than 5%, or less than 1% of the nucleicacid-cleaving activity of the wild-type exemplary endonuclease (e.g.,Cas9 from S. pyogenes, supra). The modified form of the endonuclease canhave no substantial nucleic acid-cleaving activity. When an endonucleaseis a modified form that has no substantial nucleic acid-cleavingactivity, it is referred to herein as “enzymatically inactive.”

Mutations contemplated can include substitutions, additions, anddeletions, or any combination thereof. The mutation converts the mutatedamino acid to alanine. The mutation converts the mutated amino acid toanother amino acid (e.g., glycine, serine, threonine, cysteine, valine,leucine, isoleucine, methionine, proline, phenylalanine, tyrosine,tryptophan, aspartic acid, glutamic acid, asparagine, glutamine,histidine, lysine, or arginine). The mutation converts the mutated aminoacid to a non-natural amino acid (e.g., selenomethionine). The mutationconverts the mutated amino acid to amino acid mimics (e.g.,phosphomimics). The mutation can be a conservative mutation. Forexample, the mutation converts the mutated amino acid to amino acidsthat resemble the size, shape, charge, polarity, conformation, and/orrotamers of the mutated amino acids (e.g., cysteine/serine mutation,lysine/asparagine mutation, histidine/phenylalanine mutation). Themutation can cause a shift in reading frame and/or the creation of apremature stop codon. Mutations can cause changes to regulatory regionsof genes or loci that affect expression of one or more genes.

Guide RNAs

The present disclosure provides a guide RNAs (gRNAs) that can direct theactivities of an associated endonuclease to a specific target sitewithin a polynucleotide. A guide RNA can comprise at least a spacersequence that hybridizes to a target nucleic acid sequence of interest,and a CRISPR repeat sequence. In CRISPR Type II systems, the gRNA alsocomprises a second RNA called the tracrRNA sequence. In the CRISPR TypeII guide RNA (gRNA), the CRISPR repeat sequence and tracrRNA sequencehybridize to each other to form a duplex. In CRISPR Type V systems, thegRNA comprises a crRNA that forms a duplex. In some embodiments, a gRNAcan bind an endonuclease, such that the gRNA and endonuclease form acomplex. The gRNA can provide target specificity to the complex byvirtue of its association with the endonuclease. The genome-targetingnucleic acid thus can direct the activity of the endonuclease.

Exemplary guide RNAs include a spacer sequences that comprises 15-200nucleotides wherein the gRNA targets a genome location based on theGRCh38 human genome assembly. As is understood by the person of ordinaryskill in the art, each gRNA can be designed to include a spacer sequencecomplementary to its genomic target site or region. See Jinek et al.,Science, 2012, 337, 816-821 and Deltcheva et al., Nature, 2011, 471,602-607.

The gRNA can be a double-molecule guide RNA. The gRNA can be asingle-molecule guide RNA.

A double-molecule guide RNA can comprise two strands of RNA. The firststrand comprises in the 5′ to 3′ direction, an optional spacer extensionsequence, a spacer sequence and a minimum CRISPR repeat sequence. Thesecond strand can comprise a minimum tracrRNA sequence (complementary tothe minimum CRISPR repeat sequence), a 3′ tracrRNA sequence and anoptional tracrRNA extension sequence.

A single-molecule guide RNA (sgRNA) can comprise, in the 5′ to 3′direction, an optional spacer extension sequence, a spacer sequence, aminimum CRISPR repeat sequence, a single-molecule guide linker, aminimum tracrRNA sequence, a 3′ tracrRNA sequence and an optionaltracrRNA extension sequence. The optional tracrRNA extension cancomprise elements that contribute additional functionality (e.g.,stability) to the guide RNA. The single-molecule guide linker can linkthe minimum CRISPR repeat and the minimum tracrRNA sequence to form ahairpin structure. The optional tracrRNA extension can comprise one ormore hairpins.

In some embodiments, a sgRNA comprises a 20 nucleotide spacer sequenceat the 5′ end of the sgRNA sequence. In some embodiments, a sgRNAcomprises a less than a 20 nucleotide spacer sequence at the 5′ end ofthe sgRNA sequence. In some embodiments, a sgRNA comprises a more than20 nucleotide spacer sequence at the 5′ end of the sgRNA sequence. Insome embodiments, a sgRNA comprises a variable length spacer sequencewith 17-30 nucleotides at the 5′ end of the sgRNA sequence. In someembodiments, a sgRNA comprises a spacer extension sequence with a lengthof more than 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90,100, 120, 140, 160, 180, or 200 nucleotides. In some embodiments, asgRNA comprises a spacer extension sequence with a length of less than3, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, or 100nucleotides.

In some embodiments, a sgRNA comprises a spacer extension sequence thatcomprises another moiety (e.g., a stability control sequence, anendoribonuclease binding sequence, or a ribozyme). The moiety candecrease or increase the stability of a nucleic acid targeting nucleicacid. The moiety can be a transcriptional terminator segment (i.e., atranscription termination sequence). The moiety can function in aeukaryotic cell. The moiety can function in a prokaryotic cell. Themoiety can function in both eukaryotic and prokaryotic cells.Non-limiting examples of suitable moieties include: a 5′ cap (e.g., a7-methylguanylate cap (m7 G)), a riboswitch sequence (e.g., to allow forregulated stability and/or regulated accessibility by proteins andprotein complexes), a sequence that forms a dsRNA duplex (i.e., ahairpin), a sequence that targets the RNA to a subcellular location(e.g., nucleus, mitochondria, chloroplasts, and the like), amodification or sequence that provides for tracking (e.g., directconjugation to a fluorescent molecule, conjugation to a moiety thatfacilitates fluorescent detection, a sequence that allows forfluorescent detection, etc.), and/or a modification or sequence thatprovides a binding site for proteins (e.g., proteins that act on DNA,including transcriptional activators, transcriptional repressors, DNAmethyltransferases, DNA demethylases, histone acetyltransferases,histone deacetylases, and the like).

In some embodiments, a sgRNA comprises a spacer sequence that hybridizesto a sequence in a target polynucleotide. The spacer of a gRNA caninteract with a target polynucleotide in a sequence-specific manner viahybridization (i.e., base pairing). The nucleotide sequence of thespacer can vary depending on the sequence of the target nucleic acid ofinterest.

In a CRISPR-endonuclease system, a spacer sequence can be designed tohybridize to a target polynucleotide that is located 5′ of a PAM of theendonuclease used in the system. The spacer may perfectly match thetarget sequence or may have mismatches. Each endonuclease, e.g., Cas9nuclease, has a particular PAM sequence that it recognizes in a targetDNA. For example, S. pyogenes Cas9 recognizes a PAM that comprises thesequence 5′-NRG-3′, where R comprises either A or G, where N is anynucleotide and N is immediately 3′ of the target nucleic acid sequencetargeted by the spacer sequence.

A target polynucleotide sequence can comprise 20 nucleotides. The targetpolynucleotide can comprise less than 20 nucleotides. The targetpolynucleotide can comprise more than 20 nucleotides. The targetpolynucleotide can comprise at least: 5, 10, 15, 16, 17, 18, 19, 20, 21,22, 23, 24, 25, 30 or more nucleotides. The target polynucleotide cancomprise at most: 5, 10, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30or more nucleotides. The target polynucleotide sequence can comprise 20bases immediately 5′ of the first nucleotide of the PAM.

A spacer sequence that hybridizes to a target polynucleotide can have alength of at least about 6 nucleotides (nt). The spacer sequence can beat least about 6 nt, at least about 10 nt, at least about 15 nt, atleast about 18 nt, at least about 19 nt, at least about 20 nt, at leastabout 25 nt, at least about 30 nt, at least about 35 nt or at leastabout 40 nt, from about 6 nt to about 80 nt, from about 6 nt to about 50nt, from about 6 nt to about 45 nt, from about 6 nt to about 40 nt, fromabout 6 nt to about 35 nt, from about 6 nt to about 30 nt, from about 6nt to about 25 nt, from about 6 nt to about 20 nt, from about 6 nt toabout 19 nt, from about 10 nt to about 50 nt, from about 10 nt to about45 nt, from about 10 nt to about 40 nt, from about 10 nt to about 35 nt,from about 10 nt to about 30 nt, from about 10 nt to about 25 nt, fromabout 10 nt to about 20 nt, from about 10 nt to about 19 nt, from about19 nt to about 25 nt, from about 19 nt to about 30 nt, from about 19 ntto about 35 nt, from about 19 nt to about 40 nt, from about 19 nt toabout 45 nt, from about 19 nt to about 50 nt, from about 19 nt to about60 nt, from about 20 nt to about 25 nt, from about 20 nt to about 30 nt,from about 20 nt to about 35 nt, from about 20 nt to about 40 nt, fromabout 20 nt to about 45 nt, from about 20 nt to about 50 nt, or fromabout 20 nt to about 60 nt. In some examples, the spacer sequence cancomprise 20 nucleotides. In some examples, the spacer can comprise 19nucleotides. In some examples, the spacer can comprise 18 nucleotides.In some examples, the spacer can comprise 22 nucleotides.

In some examples, the percent complementarity between the spacersequence and the target nucleic acid is at least about 30%, at leastabout 40%, at least about 50%, at least about 60%, at least about 65%,at least about 70%, at least about 75%, at least about 80%, at leastabout 85%, at least about 90%, at least about 95%, at least about 97%,at least about 98%, at least about 99%, or 100%. In some examples, thepercent complementarity between the spacer sequence and the targetnucleic acid is at most about 30%, at most about 40%, at most about 50%,at most about 60%, at most about 65%, at most about 70%, at most about75%, at most about 80%, at most about 85%, at most about 90%, at mostabout 95%, at most about 97%, at most about 98%, at most about 99%, or100%. In some examples, the percent complementarity between the spacersequence and the target nucleic acid is 100% over the six contiguous5′-most nucleotides of the target sequence of the complementary strandof the target nucleic acid. The percent complementarity between thespacer sequence and the target nucleic acid can be at least 60% overabout 20 contiguous nucleotides. The length of the spacer sequence andthe target nucleic acid can differ by 1 to 6 nucleotides, which may bethought of as a bulge or bulges.

A tracrRNA sequence can comprise nucleotides that hybridize to a minimumCRISPR repeat sequence in a cell. A minimum tracrRNA sequence and aminimum CRISPR repeat sequence may form a duplex, i.e. a base-paireddouble-stranded structure. Together, the minimum tracrRNA sequence andthe minimum CRISPR repeat can bind to an RNA-guided endonuclease. Atleast a part of the minimum tracrRNA sequence can hybridize to theminimum CRISPR repeat sequence. The minimum tracrRNA sequence can be atleast about 30%, about 40%, about 50%, about 60%, about 65%, about 70%,about 75%, about 80%, about 85%, about 90%, about 95%, or 100%complementary to the minimum CRISPR repeat sequence.

The minimum tracrRNA sequence can have a length from about 7 nucleotidesto about 100 nucleotides. For example, the minimum tracrRNA sequence canbe from about 7 nucleotides (nt) to about 50 nt, from about 7 nt toabout 40 nt, from about 7 nt to about 30 nt, from about 7 nt to about 25nt, from about 7 nt to about 20 nt, from about 7 nt to about 15 nt, fromabout 8 nt to about 40 nt, from about 8 nt to about 30 nt, from about 8nt to about 25 nt, from about 8 nt to about 20 nt, from about 8 nt toabout 15 nt, from about 15 nt to about 100 nt, from about 15 nt to about80 nt, from about 15 nt to about 50 nt, from about 15 nt to about 40 nt,from about 15 nt to about 30 nt or from about 15 nt to about 25 nt long.The minimum tracrRNA sequence can be approximately 9 nucleotides inlength. The minimum tracrRNA sequence can be approximately 12nucleotides. The minimum tracrRNA can consist of tracrRNA nt 23-48described in Jinek et al., supra.

The minimum tracrRNA sequence can be at least about 60% identical to areference minimum tracrRNA (e.g., wild type, tracrRNA from S. pyogenes)sequence over a stretch of at least 6, 7, or 8 contiguous nucleotides.For example, the minimum tracrRNA sequence can be at least about 65%identical, about 70% identical, about 75% identical, about 80%identical, about 85% identical, about 90% identical, about 95%identical, about 98% identical, about 99% identical or 100% identical toa reference minimum tracrRNA sequence over a stretch of at least 6, 7,or 8 contiguous nucleotides.

The duplex between the minimum CRISPR RNA and the minimum tracrRNA cancomprise a double helix. The duplex between the minimum CRISPR RNA andthe minimum tracrRNA can comprise at least about 1, 2, 3, 4, 5, 6, 7, 8,9, or 10 or more nucleotides. The duplex between the minimum CRISPR RNAand the minimum tracrRNA can comprise at most about 1, 2, 3, 4, 5, 6, 7,8, 9, or 10 or more nucleotides.

The duplex can comprise a mismatch (i.e., the two strands of the duplexare not 100% complementary). The duplex can comprise at least about 1,2, 3, 4, or 5 or mismatches. The duplex can comprise at most about 1, 2,3, 4, or 5 or mismatches. The duplex can comprise no more than 2mismatches.

In some embodiments, a tracrRNA may be a 3′ tracrRNA. In someembodiments, a 3′ tracrRNA sequence can comprise a sequence with atleast about 30%, about 40%, about 50%, about 60%, about 65%, about 70%,about 75%, about 80%, about 85%, about 90%, about 95%, or 100% sequenceidentity to a reference tracrRNA sequence (e.g., a tracrRNA from S.pyogenes).

In some embodiments, a gRNA may comprise a tracrRNA extension sequence.A tracrRNA extension sequence can have a length from about 1 nucleotideto about 400 nucleotides. The tracrRNA extension sequence can have alength of more than 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70,80, 90, 100, 120, 140, 160, 180, or 200 nucleotides. The tracrRNAextension sequence can have a length from about 20 to about 5000 or morenucleotides. The tracrRNA extension sequence can have a length of lessthan 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, or 100nucleotides. The tracrRNA extension sequence can comprise less than 10nucleotides in length. The tracrRNA extension sequence can be 10-30nucleotides in length. The tracrRNA extension sequence can be 30-70nucleotides in length.

The tracrRNA extension sequence can comprise a functional moiety (e.g.,a stability control sequence, ribozyme, endoribonuclease bindingsequence). The functional moiety can comprise a transcriptionalterminator segment (i.e., a transcription termination sequence). Thefunctional moiety can have a total length from about 10 nucleotides (nt)to about 100 nucleotides, from about 10 nt to about 20 nt, from about 20nt to about 30 nt, from about 30 nt to about 40 nt, from about 40 nt toabout 50 nt, from about 50 nt to about 60 nt, from about 60 nt to about70 nt, from about 70 nt to about 80 nt, from about 80 nt to about 90 nt,or from about 90 nt to about 100 nt, from about 15 nt to about 80 nt,from about 15 nt to about 50 nt, from about 15 nt to about 40 nt, fromabout 15 nt to about 30 nt, or from about 15 nt to about 25 nt.

In some embodiments, a sgRNA may comprise a linker sequence with alength from about 3 nucleotides to about 100 nucleotides. In Jinek etal., supra, for example, a simple 4 nucleotide “tetraloop” (-GAAA-) wasused (Jinek et al., Science, 2012, 337(6096):816-821). An illustrativelinker has a length from about 3 nucleotides (nt) to about 90 nt, fromabout 3 nt to about 80 nt, from about 3 nt to about 70 nt, from about 3nt to about 60 nt, from about 3 nt to about 50 nt, from about 3 nt toabout 40 nt, from about 3 nt to about 30 nt, from about 3 nt to about 20nt, from about 3 nt to about 10 nt. For example, the linker can have alength from about 3 nt to about 5 nt, from about 5 nt to about 10 nt,from about 10 nt to about 15 nt, from about 15 nt to about 20 nt, fromabout 20 nt to about 25 nt, from about 25 nt to about 30 nt, from about30 nt to about 35 nt, from about 35 nt to about 40 nt, from about 40 ntto about 50 nt, from about 50 nt to about 60 nt, from about 60 nt toabout 70 nt, from about 70 nt to about 80 nt, from about 80 nt to about90 nt, or from about 90 nt to about 100 nt. The linker of asingle-molecule guide nucleic acid can be between 4 and 40 nucleotides.The linker can be at least about 100, 500, 1000, 1500, 2000, 2500, 3000,3500, 4000, 4500, 5000, 5500, 6000, 6500, or 7000 or more nucleotides.The linker can be at most about 100, 500, 1000, 1500, 2000, 2500, 3000,3500, 4000, 4500, 5000, 5500, 6000, 6500, or 7000 or more nucleotides.

Linkers can comprise any of a variety of sequences, although in someexamples the linker will not comprise sequences that have extensiveregions of homology with other portions of the guide RNA, which mightcause intramolecular binding that could interfere with other functionalregions of the guide. In Jinek et al., supra, a simple 4 nucleotidesequence -GAAA- was used (Jinek et al., Science, 2012,337(6096):816-821), but numerous other sequences, including longersequences can likewise be used.

The linker sequence can comprise a functional moiety. For example, thelinker sequence can comprise one or more features, including an aptamer,a ribozyme, a protein-interacting hairpin, a protein binding site, aCRISPR array, an intron, or an exon. The linker sequence can comprise atleast about 1, 2, 3, 4, or 5 or more functional moieties. In someexamples, the linker sequence can comprise at most about 1, 2, 3, 4, or5 or more functional moieties.

In some embodiments, a sgRNA does not comprise a uracil, e.g., at the3′end of the sgRNA sequence. In some embodiments, a sgRNA does compriseone or more uracils, e.g., at the 3′end of the sgRNA sequence. In someembodiments, a sgRNA comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 uracils(U) at the 3′ end of the sgRNA sequence.

A sgRNA may be chemically modified. In some embodiments, a chemicallymodified gRNA is a gRNA that comprises at least one nucleotide with achemical modification, e.g., a 2′-O-methyl sugar modification. In someembodiments, a chemically modified gRNA comprises a modified nucleicacid backbone. In some embodiments, a chemically modified gRNA comprisesa 2′-O-methyl-phosphorothioate residue. In some embodiments, chemicalmodifications enhance stability, reduce the likelihood or degree ofinnate immune response, and/or enhance other attributes, as described inthe art.

In some embodiments, a modified gRNA may comprise a modified backbones,for example, phosphorothioates, phosphotriesters, morpholinos, methylphosphonates, short chain alkyl or cycloalkyl intersugar linkages orshort chain heteroatomic or heterocyclic intersugar linkages.

Morpholino-based compounds are described in Braasch and David Corey,Biochemistry, 2002, 41(14): 4503-4510; Genesis, 2001, Volume 30, Issue3; Heasman, Dev. Biol., 2002, 243: 209-214; Nasevicius et al., Nat.Genet., 2000, 26:216-220; Lacerra et al., Proc. Natl. Acad. Sci., 2000,97: 9591-9596.; and U.S. Pat. No. 5,034,506, issued Jul. 23, 1991.

Cyclohexenyl nucleic acid oligonucleotide mimetics are described in Wanget al., J. Am. Chem. Soc., 2000, 122: 8595-8602.

In some embodiments, a modified gRNA may comprise one or moresubstituted sugar moieties, e.g., one of the following at the 2′position: OH, SH, SCH₃, F, OCN, OCH₃, OCH₃ O(CH₂)n CH₃, O(CH₂)n NH₂, orO(CH₂)n CH₃, where n is from 1 to about 10; C1 to C10 lower alkyl,alkoxyalkoxy, substituted lower alkyl, alkaryl or aralkyl; Cl; Br; CN;CF₃; OCF₃; O-, S-, or N-alkyl; O-, S-, or N-alkenyl; SOCH₃; SO₂ CH₃;ONO₂; NO₂; N₃; NH₂; heterocycloalkyl; heterocycloalkaryl;aminoalkylamino; polyalkylamino; substituted silyl; an RNA cleavinggroup; a reporter group; an intercalator; 2′-O-(2-methoxyethyl);2′-methoxy (2′-O—CH₃); 2′-propoxy (2′-OCH₂ CH₂CH₃); and 2′-fluoro(2′-F). Similar modifications may also be made at other positions on thegRNA, particularly the 3′ position of the sugar on the 3′ terminalnucleotide and the 5′ position of 5′ terminal nucleotide. In someexamples, both a sugar and an internucleoside linkage, i.e., thebackbone, of the nucleotide units can be replaced with novel groups.

Guide RNAs can also include, additionally or alternatively, nucleobase(often referred to in the art simply as “base”) modifications orsubstitutions. As used herein, “unmodified” or “natural” nucleobasesinclude adenine (A), guanine (G), thymine (T), cytosine (C), and uracil(U). Modified nucleobases include nucleobases found only infrequently ortransiently in natural nucleic acids, e.g., hypoxanthine,6-methyladenine, 5-Me pyrimidines, particularly 5-methylcytosine (alsoreferred to as 5-methyl-2′ deoxycytosine and often referred to in theart as 5-Me-C), 5-hydroxymethylcytosine (HMC), glycosyl HMC andgentobiosyl HMC, as well as synthetic nucleobases, e.g., 2-aminoadenine,2-(methylamino)adenine, 2-(imidazolylalkyl)adenine,2-(aminoalklyamino)adenine or other heterosubstituted alkyladenines,2-thiouracil, 2-thiothymine, 5-bromouracil, 5-hydroxymethyluracil,8-azaguanine, 7-deazaguanine, N6 (6-aminohexyl)adenine, and2,6-diaminopurine. Kornberg, A., DNA Replication, W. H. Freeman & Co.,San Francisco, pp 75-′7′7, 1980; Gebeyehu et al., Nucl. Acids Res. 1997,15:4513. A “universal” base known in the art, e.g., inosine, can also beincluded. 5-Me-C substitutions have been shown to increase nucleic acidduplex stability by 0.6-1.2° C. (Sanghvi, Y. S., in Crooke, S. T. andLebleu, B., eds., Antisense Research and Applications, CRC Press, BocaRaton, 1993, pp. 276-278) and are aspects of base substitutions.

Modified nucleobases can comprise other synthetic and naturalnucleobases, such as 5-methylcytosine (5-me-C), 5-hydroxymethylcytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and otheralkyl derivatives of adenine and guanine, 2-propyl and other alkylderivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and2-thiocytosine, 5-halouracil and cytosine, 5-propynyl uracil andcytosine, 6-azo uracil, cytosine and thymine, 5-uracil (pseudo-uracil),4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl andother 8-substituted adenines and guanines, 5-halo particularly 5-bromo,5-trifluoromethyl and other 5-substituted uracils and cytosines,7-methylquanine and 7-methyladenine, 8-azaguanine and 8-azaadenine,7-deazaguanine and 7-deazaadenine, and 3-deazaguanine and3-deazaadenine.

Complexes of a Genome-Targeting Nucleic Acid and a Endonuclease

A gRNA interacts with an endonuclease (e.g., a RNA-guided nuclease suchas Cas9), thereby forming a complex. The gRNA guides the endonuclease toa target polynucleotide.

The endonuclease and gRNA can each be administered separately to a cellor a subject. In some embodiments, the endonuclease can be pre-complexedwith one or more guide RNAs, or one or more crRNA together with atracrRNA. The pre-complexed material can then be administered to a cellor a subject. Such pre-complexed material is known as aribonucleoprotein particle (RNP). The endonuclease in the RNP can be,for example, a Cas9 endonuclease or a Cpf1 endonuclease. Theendonuclease can be flanked at the N-terminus, the C-terminus, or boththe N-terminus and C-terminus by one or more nuclear localizationsignals (NLSs). For example, a Cas9 endonuclease can be flanked by twoNLSs, one NLS located at the N-terminus and the second NLS located atthe C-terminus. The NLS can be any NLS known in the art, such as a SV40NLS. The weight ratio of genome-targeting nucleic acid to endonucleasein the RNP can be 1:1. For example, the weight ratio of sgRNA to Cas9endonuclease in the RNP can be 1:1.

Nucleic Acids Encoding System Components

The present disclosure provides a nucleic acid comprising a nucleotidesequence encoding a genome-targeting nucleic acid of the disclosure, anendonuclease of the disclosure, and/or any nucleic acid or proteinaceousmolecule necessary to carry out the aspects of the methods of thedisclosure. The encoding nucleic acids can be RNA, DNA, or a combinationthereof.

The nucleic acid encoding a genome-targeting nucleic acid of thedisclosure, an endonuclease of the disclosure, and/or any nucleic acidor proteinaceous molecule necessary to carry out the aspects of themethods of the disclosure can comprise a vector (e.g., a recombinantexpression vector).

The term “vector” refers to a nucleic acid molecule capable oftransporting another nucleic acid to which it has been linked. One typeof vector is a “plasmid”, which refers to a circular double-stranded DNAloop into which additional nucleic acid segments can be ligated. Anothertype of vector is a viral vector, wherein additional nucleic acidsegments can be ligated into the viral genome. Certain vectors arecapable of autonomous replication in a host cell into which they areintroduced (e.g., bacterial vectors having a bacterial origin ofreplication and episomal mammalian vectors). Other vectors (e.g.,non-episomal mammalian vectors) are integrated into the genome of a hostcell upon introduction into the host cell, and thereby are replicatedalong with the host genome.

In some examples, vectors can be capable of directing the expression ofnucleic acids to which they are operatively linked. Such vectors arereferred to herein as “recombinant expression vectors”, or more simply“expression vectors”, which serve equivalent functions.

The term “operably linked” means that the nucleotide sequence ofinterest is linked to regulatory sequence(s) in a manner that allows forexpression of the nucleotide sequence. The term “regulatory sequence” isintended to include, for example, promoters, enhancers and otherexpression control elements (e.g., polyadenylation signals). Suchregulatory sequences are well known in the art and are described, forexample, in Goeddel; Gene Expression Technology: Methods in Enzymology,1990, 185, Academic Press, San Diego, Calif. Regulatory sequencesinclude those that direct constitutive expression of a nucleotidesequence in many types of host cells, and those that direct expressionof the nucleotide sequence only in certain host cells (e.g.,tissue-specific regulatory sequences). It will be appreciated by thoseskilled in the art that the design of the expression vector can dependon such factors as the choice of the target cell, the level ofexpression desired, and the like.

Expression vectors contemplated include, but are not limited to, viralvectors based on vaccinia virus, poliovirus, adenovirus,adeno-associated virus, SV40, herpes simplex virus, humanimmunodeficiency virus, retrovirus (e.g., Murine Leukemia Virus, spleennecrosis virus, and vectors derived from retroviruses such as RousSarcoma Virus, Harvey Sarcoma Virus, avian leukosis virus, a lentivirus,human immunodeficiency virus, myeloproliferative sarcoma virus, andmammary tumor virus) and other recombinant vectors. Other vectorscontemplated for eukaryotic target cells include, but are not limitedto, the vectors pXT1, pSG5, pSVK3, pBPV, pMSG, and pSVLSV40 (Pharmacia).Other vectors can be used so long as they are compatible with the hostcell.

In some examples, a vector can comprise one or more transcription and/ortranslation control elements. Depending on the host/vector systemutilized, any of a number of suitable transcription and translationcontrol elements, including constitutive and inducible promoters,transcription enhancer elements, transcription terminators, etc. can beused in the expression vector. The vector can be a self-inactivatingvector that either inactivates the viral sequences or the components ofthe CRISPR machinery or other elements.

Non-limiting examples of suitable eukaryotic promoters (i.e., promotersfunctional in a eukaryotic cell) include those from cytomegalovirus(CMV) immediate early, herpes simplex virus (HSV) thymidine kinase,early and late SV40, long terminal repeats (LTRs) from retrovirus, humanelongation factor-1 promoter (EF1), chicken beta-actin promoter (CBA),ubiquitin C promoter (UBC), a hybrid construct comprising thecytomegalovirus enhancer fused to the chicken beta-actin promoter (CAG),a hybrid construct comprising the cytomegalovirus enhancer fused to thepromoter, the first exon, and the first intron of chicken beta-actingene (CAG or CAGGS), murine stem cell virus promoter (MSCV),phosphoglycerate kinase-1 locus promoter (PGK), and mousemetallothionein-I promoter.

A promoter can be an inducible promoter (e.g., a heat shock promoter,tetracycline-regulated promoter, steroid-regulated promoter,metal-regulated promoter, estrogen receptor-regulated promoter, etc.).The promoter can be a constitutive promoter (e.g., CMV promoter, UBCpromoter, CAG promoter). In some cases, the promoter can be a spatiallyrestricted and/or temporally restricted promoter (e.g., a tissuespecific promoter, a cell type specific promoter, etc.).

Introduction of the complexes, polypeptides, and nucleic acids of thedisclosure into cells can occur by viral or bacteriophage infection,transfection, conjugation, protoplast fusion, lipofection,electroporation, nucleofection, calcium phosphate precipitation,polyethyleneimine (PEI)-mediated transfection, DEAE-dextran mediatedtransfection, liposome-mediated transfection, particle gun technology,calcium phosphate precipitation, direct micro-injection,nanoparticle-mediated nucleic acid delivery, and the like.

III. Strategies to Evade Immune Response and Increase Survival

Described herein are strategies to enable genetically modified cells,i.e., universal donor cells, to evade immune response and/or increasetheir survival, or viability following engraftment into a subject. Insome embodiments, these strategies enable universal donor cells to evadeimmune response and/or survive at higher success rates than anunmodified cell. In some embodiments, genetically modified cellscomprise the introduction of at least one genetic modification within ornear at least one gene that decreases the expression of one or moreMHC-I and MHC-II human leukocyte antigens relative to an unmodifiedcell; at least one genetic modification that increases the expression ofat least one polynucleotide that encodes a tolerogenic factor relativeto an unmodified cell; and/or at least one genetic modification thatalters the expression of at least one gene that encodes a survivalfactor relative to an unmodified cell. In some embodiments, geneticallymodified cells comprise the introduction of at least one geneticmodification within or near at least one gene that decreases theexpression of one or more MHC-I and MHC-II human leukocyte antigensrelative to an unmodified cell; at least one genetic modification thatincreases the expression of at least one polynucleotide that encodes atolerogenic factor relative to an unmodified cell; and at least onegenetic modification that alters the expression of at least one genethat encodes a survival factor relative to an unmodified cell. In otherembodiments, genetically modified cells comprise at least one deletionor insertion-deletion mutation within or near at least one gene thatalters the expression of one or more MHC-I and MHC-II human leukocyteantigens relative to an unmodified cell; and at least one insertion of apolynucleotide that encodes at least one tolerogenic factor at a sitethat partially overlaps, completely overlaps, or is contained within,the site of a deletion of a gene that alters the expression of one ormore MHC-I and MHC-II HLAs. In yet other embodiments, geneticallymodified cells comprise at least one genetic modification that altersthe expression of at least one gene that encodes a survival factorrelative to an unmodified cell.

The genes that encode the major histocompatibility complex (MHC) arelocated on human Chr. 6p21. The resultant proteins coded by the WICgenes are a series of surface proteins that are essential in donorcompatibility during cellular transplantation. WIC genes are dividedinto WIC class I (MHC-I) and MHC class II (MHC-II). MHC-I genes (HLA-A,HLA-B, and HLA-C) are expressed in almost all tissue cell types,presenting “non-self” antigen-processed peptides to CD8+ T cells,thereby promoting their activation to cytolytic CD8+ T cells.Transplanted or engrafted cells expressing “non-self” MHC-I moleculeswill cause a robust cellular immune response directed at these cells andultimately resulting in their demise by activated cytolytic CD8+ Tcells. MHC-I proteins are intimately associated withbeta-2-microglobulin (B2M) in the endoplasmic reticulum, which isessential for forming functional MHC-I molecules on the cell surface. Inaddition, there are three non-classical MHC-Ib molecules (HLA-E, HLA-F,and HLA-G), which have immune regulatory functions. MHC-II biomoleculeinclude HLA-DP, HLA-DM, HLA-DOA, HLA-DOB, HLA-DQ, and HLA-DR. Due totheir primary function in the immune response, MHC-I and MHC-IIbiomolecules contribute to immune rejection following cellularengraftment of non-host cells, e.g., cellular engraftment for purposesof regenerative medicine.

MHC-I cell surface molecules are composed of MHC-encoded heavy chains(HLA-A, HLA-B, or HLA-C) and the invariant subunit beta-2-microglobulin(B2M). Thus, a reduction in the concentration of B2M within a cellallows for an effective method of reducing the cell surface expressionof MHC-I cell surface molecules.

In some embodiments, a cell comprises a genomic modification of one ormore MHC-I or MHC-II genes. In some embodiments, a cell comprises agenomic modification of one or more polynucleotide sequences thatregulates the expression of MHC-I and/or MHC-II. In some embodiments, agenetic modification of the disclosure is performed using any geneediting method including but not limited to those methods describedherein.

In some embodiments, decreasing the expression of one or more MHC-I andMHC-II human leukocyte antigens relative to an unmodified cell isaccomplished by targeting, e.g., for genetic deletion and/or insertionof at least one base pair, in a MHC-I and/or MHC-II gene directly. Insome embodiments, decreasing the expression of one or more MHC-I andMHC-II human leukocyte antigens relative to an unmodified cell isaccomplished by targeting, e.g., for genetic deletion, a CIITA gene. Insome embodiments, decreasing the expression of one or more MHC-I andMHC-II human leukocyte antigens relative to an unmodified cell isaccomplished by targeting, e.g., for genetic deletion, at least onetranscriptional regulator of MHC-I or MHC-II. In some embodiments, atranscriptional regulator of MHC-I or MHC-II is a NLRC5, or CIITA gene.In some embodiments, a transcriptional regulator of MHC-I or MHC-II is aRFX5, RFXAP, RFXANK, NFY-A, NFY-B, NFY-C, IRF-1, and/or TAP1 gene.

In some embodiments, the genome of a cell has been modified to deletethe entirety or a portion of a HLA-A, HLA-B, and/or HLA-C gene. In someembodiments, the genome of a cell has been modified to delete theentirety or a portion of a promoter region of a HLA-A, HLA-B, and/orHLA-C gene. In some embodiments, the genome of a cell has been modifiedto delete the entirety or a portion of a gene that encodes atranscriptional regulator of MHC-I or MHC-II. In some embodiments, thegenome of a cell has been modified to delete the entirety or a portionof a promoter region of a gene that encodes a transcriptional regulatorof MHC-I or MHC-II.

In some embodiments, the genome of a cell has been modified to decreasethe expression of beta-2-microglobulin (B2M). B2M is a non-polymorphicgene that encodes a common protein subunit required for surfaceexpression of all polymorphic MEW class I heavy chains. HLA-I proteinsare intimately associated with B2M in the endoplasmic reticulum, whichis essential for forming functional, cell-surface expressed HLA-Imolecules. In some embodiments, the gRNA targets a site within the B2Mgene comprising a 5′ GCTACTCTCTCTTTCTGGCC 3′ sequence (SEQ ID NO: 1). Insome embodiments, the gRNA targets a site within the B2M gene comprisinga 5′ GGCCGAGATGTCTCGCTCCG 3′ sequence (SEQ ID NO: 2). In someembodiments, the gRNA targets a site within the B2M gene comprising a 5′CGCGAGCACAGCTAAGGCCA 3′ sequence (SEQ ID NO: 3). In alternateembodiments, the gRNA targets a site within the B2M gene comprising anyof the following sequences: 5′-TATAAGTGGAGGCGTCGCGC-3′ (SEQ ID NO: 35),5′-GAGTAGCGCGAGCACAGCTA-3′ (SEQ ID NO: 36), 5′-ACTGGACGCGTCGCGCTGGC-3′(SEQ ID NO: 37), 5′-AAGTGGAGGCGTCGCGCTGG-3′ (SEQ ID NO: 38),5-GGCCACGGAGCGAGACATCT-3′ (SEQ ID NO: 39), 5′-GCCCGAATGCTGTCAGCTTC-3′(SEQ ID NO: 40). 5′-CTCGCGCTACTCTCTCTTTC-3′ (SEQ ID NO: 41),5′-TCCTGAAGCTGACAGCATTC-3′ (SEQ ID NO: 42), 5′-TTCCTGAAGCTGACAGCATT-3′(SEQ ID NO: 43), or 5′-ACTCTCTCTTTCTGGCCTGG-3′ (SEQ ID NO: 44). In someembodiments, the gRNA comprises a polynucleotide sequence of any one ofSEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 35, SEQ ID NO: 36,SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO:41, SEQ ID NO: 42, SEQ ID NO: 43, or SEQ ID NO: 44. The gRNA/CRISPRnuclease complex targets and cleaves a target site in the B2M locus.Repair of a double-stranded break by NHEJ can result in a deletion of atleast on nucleotide and/or an insertion of at least one nucleotide,thereby disrupting or eliminating expression of B2M. Alternatively, theB2M locus can be targeted by at least two CRISPR systems each comprisinga different gRNA, such that cleavage at two sites in the B2M locus leadsto a deletion of the sequence between the two cuts, thereby eliminatingexpression of B2M.

In some embodiments, the genome of a cell has been modified to decreasethe expression of Class II transactivator (CIITA). CIITA is a member ofthe LR or nucleotide binding domain (NBD) leucine-rich repeat (LRR)family of proteins and regulates the transcription of by associatingwith the WIC enhanceosome. The expression of CIITA is induced in B cellsand dendritic cells as a function of developmental stage and isinducible by IFN-γ in most cell types.

In some embodiments, the genome of a cell has been modified to decreasethe expression of the NLR family, CARD domain containing 5 (NLRC5).NLRC5 is a critical regulator of MHC-I-mediated immune responses and,similar to CIITA, NLRC5 is highly inducible by IFN-γ and can translocateinto the nucleus. NLRC5 activates the promoters of MHC-I genes andinduces the transcription of MHC-I as well as related genes involved inMHC-I antigen presentation.

In some embodiments, tolerogenic factors can be inserted or reinsertedinto genetically modified cells to create immune-privileged universaldonor cells. In some embodiments, the universal donor cells disclosedherein have been further modified to express one or more tolerogenicfactors. Exemplary tolerogenic factors include, without limitation, oneor more of HLA-C, HLA-E, HLA-F, HLA-G, PD-L1, CTLA-4-Ig, CD47,CI-inhibitor, and IL-35. In some embodiments, the genetic modification,e.g., insertion, of at least one polynucleotide encoding at least onetolerogenic factor enables a universal donor cell to inhibit or evadeimmune rejection with rates at least 1.05, at least 1.1, at least 1.25,at least 1.5, at least 2, at least 3, at least 4, at least 5, at least10, at least 20, or at least 50 times higher than an unmodified cellfollowing engraftment. In some embodiments, an insertion of apolynucleotide that encodes HLA-E, HLA-G, CTLA-4, CD47, and/or PD-L1enables a universal donor cell to inhibit or evade immune rejectionafter transplantation or engraftment into a host subject.

The polynucleotide encoding the tolerogenic factor generally comprisesleft and right homology arms that flank the sequence encoding thetolerogenic factor. The homology arms have substantial sequence homologyto genomic DNA at or near the targeted insertion site. For example, theleft homology arm can be a nucleotide sequence homologous with a regionlocated to the left or upstream of the target site or cut site and theright homology arm can be a nucleotide sequence homologous with a regionlocated to the right or downstream of the target site or cut site. Theproximal end of each homology arm can be homologous to genomic DNAsequence abutting the cut site. Alternatively, the proximal end of eachhomology arm can be homologous to genomic DNA sequence located up toabout 10, 20, 30, 40, 50, 60, or 70 nucleobases away from the cut site.As such, the polynucleotide encoding the tolerogenic factor can beinserted into the targeted gene locus within about 10, 20, 30, 40, 50,60, or 70 base pairs of the cut site, and additional genomic DNAbordering the cut site (and having no homology to a homolog arm) can bedeleted. The homology arms can range in length from about 50 nucleotidesto several of thousands of nucleotides. In some embodiments, thehomology arms can range in length from about 500 nucleotides to about1000 nucleotides. The substantial sequence homology between the homologyarms and the genomic DNA can be at least about 80%, at least about 85%,at least about 90%, at least about 95%, or at least about 99%.

In some embodiments, the homology arms are used with B2M guides (e.g.,gRNAs comprising a nucleotide sequence of SEQ ID NO: 1-3 or 35-44). Insome embodiments, the homology arms are designed to be used with any B2Mguide that would eliminate the start site of the B2M gene. In someembodiments, the B2M homology arms can comprise or consist essentiallyof a polynucleotide sequence of SEQ ID NO: 13 or 19, or a polynucleotidesequence having at least 85%, 90%, 95%, or 99% sequence identity withthat of SEQ ID NO: 13 or 19. In some embodiments, the left B2M homologyarm can comprise or consist essentially of SEQ ID NO: 13, or apolynucleotide sequence having at least 85%, 90%, 95%, or 99% sequenceidentity with that of SEQ ID NO: 13. In some embodiments, the right B2Mhomology arm can comprise or consist essentially of SEQ ID NO: 19, or apolynucleotide sequence having at least 85%, 90%, 95%, or 99% sequenceidentity with that of SEQ ID NO: 19.

The at least one polynucleotide encoding at least one tolerogenic factorcan be operably linked to an exogenous promoter. The exogenous promotercan be a constitutive, inducible, temporal-, tissue-, or celltype-specific promoter. In some embodiments, the exogenous promoter is aCMV, EF1a, PGK, CAG, or UBC promoter.

In some embodiments, the at least one polynucleotide encoding at leastone tolerogenic factor is inserted into a safe harbor locus, e.g., theAAVS 1 locus. In some embodiments, the at least one polynucleotideencoding at least one tolerogenic factor is inserted into a site orregion of genomic DNA that partially overlaps, completely overlaps, oris contained within (i.e., is within or near) a MHC-I gene, MHC-II gene,or a transcriptional regulator of MHC-I or MHC-II.

In some embodiments, a polynucleotide encoding PD-L1 is inserted at asite within or near a B2M gene locus. In some embodiments, apolynucleotide encoding PD-L1 is inserted at a site within or near a B2Mgene locus concurrent with, or following a deletion of all or part of aB2M gene or promoter. The polynucleotide encoding PD-L1 is operablylinked to an exogenous promoter. The exogenous promoter can be a CMVpromoter.

In some embodiments, a polynucleotide encoding HLA-E is inserted at asite within or near a B2M gene locus. In some embodiments, apolynucleotide encoding HLA-E is inserted at a site within or near a B2Mgene locus concurrent with, or following a deletion of all or part of aB2M gene or promoter. The polynucleotide encoding HLA-E is operablylinked to an exogenous promoter. The exogenous promoter can be a CMVpromoter.

In some embodiments, a polynucleotide encoding HLA-G is inserted at asite within or near a HLA-A, HLA-B, or HLA-C gene locus. In someembodiments, a polynucleotide encoding HLA-G is inserted at a sitewithin or near a HLA-A, HLA-B, or HLA-C gene locus concurrent with, orfollowing a deletion of a HLA-A, HLA-B, or HLA-C gene or promoter.

In some embodiments, a polynucleotide encoding CD47 is inserted at asite within or near a CIITA gene locus. In some embodiments, apolynucleotide encoding CD47 is inserted at a site within or near aCIITA gene locus concurrent with, or following a deletion of a CIITAgene or promoter.

In some embodiments, a polynucleotide encoding HLA-G is inserted at asite within or near a HLA-A, HLA-B, or HLA-C gene locus concurrent withinsertion of a polynucleotide encoding CD47 at a site within or near aCIITA gene locus.

In some embodiments, a cell comprises increased or decreased expressionof one or more survival factors. In some embodiments, a cell comprisesan insertion of one or more polynucleotide sequences that encodes asurvival factor. In some embodiments, a cell comprises a deletion of oneof more survival factors. In some embodiments, a genetic modification ofthe disclosure is performed using any gene editing method including butnot limited to those methods described herein. In some embodiments, acell comprises increased or decreased expression of at least onesurvival factor relative to an unmodified cell. In some embodiments, asurvival factor is a member or a critical pathway involved in cellsurvival, e.g., hypoxia, reactive oxygen species, nutrient deprivation,and/or oxidative stress. In some embodiments, the genetic modificationof at least one survival factor enables a universal donor cell tosurvive for a longer time period, e.g., at least 1.05, at least 1.1, atleast 1.25, at least 1.5, at least 2, at least 3, at least 4, at least5, at least 10, at least 20, or at least 50 times longer time period,than an unmodified cell following engraftment. In some embodiments, asurvival factor is ZNF143, TXNIP, FOXO1, JNK, or MANF.

In some embodiments, a cell comprises an insertion of a polynucleotidethat encodes MANF enables a universal donor cell to survive aftertransplantation or engraftment into a host subject at higher survivalrates relative to an unmodified cell. In some embodiments, apolynucleotide that encodes MANF is inserted into a safe harbor locus.In some embodiments, a polynucleotide that encodes MANF is inserted intoa gene belonging to a MHC-I, MHC-II, or transcriptional regulator ofMHC-I or

In some embodiments, the genome of a cell has been modified to deletethe entirety or a portion of a ZNF143, TXNIP, FOXO1, and/or INK gene. Insome embodiments, the genome of a cell has been modified to delete theentirety or a portion of a promoter region of a ZNF143, TXNIP, FOXO1,and/or JNK gene.

In some embodiments, more than one survival factor is geneticallymodified within a cell.

In certain embodiments, cells having no MHC-II expression and moderateexpression of MHC-I are genetically modified to have no surfaceexpression of MHC-I or MHC-II. In another embodiment, cells with nosurface expression of MHC-I/II are further edited to have expression ofPD-L1, e.g., insertion of a polynucleotide encoding PD-L1. In yetanother embodiment, cells with no surface expression of MHC-I/II arefurther edited to have expression of PD-L1, e.g., insertion of apolynucleotide encoding PD-L1, and are also genetically modified toincrease or decrease the expression of at least one gene that encodes asurvival factor relative to an unmodified cell.

In some embodiments, the cells further comprise increased or decreasedexpression, e.g., by a genetic modification, of one or more additionalgenes that are not necessarily implicated in either immune evasion orcell survival post-engraftment. In some embodiments, the cells furthercomprise increased expression of one or more safety switch proteinsrelative to an unmodified cell. In some embodiments, the cells compriseincreased expression of one or more additional genes that encode asafety switch protein. In some embodiments, a safety switch is also asuicide gene. In some embodiments, a safety switch is herpes simplexvirus-1 thymidine kinase (HSV-tk) or inducible caspase-9. In someembodiments, a polynucleotide that encodes at least one safety switch isinserted into a genome, e.g., into a safe harbor locus. In some otherembodiments, the one or more additional genes that are geneticallymodified encode one or more of safety switch proteins; targetingmodalities; receptors; signaling molecules; transcription factors;pharmaceutically active proteins or peptides; drug target candidates;and proteins promoting engraftment, trafficking, homing, viability,self-renewal, persistence, and/or survival thereof integrated with theconstruct.

One aspect of the present invention provides a method of generatinggenome-engineered universal donor cells, wherein a universal donor cellcomprises at least one targeted genomic modification at one or moreselected sites in genome, the method comprising genetically engineeringa cell type as described herein by introducing into said cells one ormore construct of to allow targeted modification at selected site;introducing into said cells one or more double strand breaks at theselected sites using one or more endonuclease capable of selected siterecognition; and culturing the edited cells to allow endogenous DNArepair to generate targeted insertions or deletions at the selectedsites; thereby obtaining genome-modified universal donor cells. Theuniversal donor cells generated by this method will comprise at leastone functional targeted genomic modification, and wherein thegenome-modified cells, if they are stem cells, are then capable of beingdifferentiated into progenitor cells or fully-differentiated cells.

In some other embodiments, the genome-engineered universal donor cellscomprise introduced or increased expression in at least one of HLA-E,HLA-G, CD47, or D-L1. In some embodiments, the genome-engineereduniversal donor cells are HLA class I and/or class II deficient. In someembodiment, the genome-engineered universal donor cells comprise B2Mnull or low. In some embodiments, the genome-engineered universal donorcells comprise integrated or non-integrated exogenous polynucleotideencoding one or more of HLA-E, HLA-G, and PD-L1 proteins. In someembodiments, said introduced expression is an increased expression fromeither non-expressed or lowly expressed genes comprised in said cells.In some embodiments, the non-integrated exogenous polynucleotides areintroduced using Sendai virus, AAV, episomal, or plasmid. In someembodiment, the universal donor cells are B2M null, with introducedexpression of one or more of HLA-E, HLA-G, PD-L1, and increased ordecreased expression of at least one safety switch protein. In anotherembodiment, the universal donor cells are HLA-A, HLA-B, and HLA-C null,with introduced expression of one or more of HLA-E, HLA-G, PD-L1, and atleast one safety switch protein. In some embodiment, the universal donorcells are B2M null, with introduced expression of one or more of HLA-E,HLA-G PD-L1, and increased or decreased expression of at least onesurvival factor, e.g., MANF. Methods of generating any of thegenetically modified cells described herein are contemplated to beperformed using at least any of the gene editing methods describedherein.

IV. Cell Types

Cells as described herein, e.g., universal donor cells (andcorresponding unmodified cells) may belong to any possible class of celltype. In some embodiments, a cell, e.g., universal donor cell (andcorresponding unmodified cell) may be a mammalian cell. In someembodiments, a cell, e.g., universal donor cell (and correspondingunmodified cell) may be a human cell. In some embodiments, a cell, e.g.,universal donor cell (and corresponding unmodified cell) may be a stemcell. In some embodiments, a cell, e.g., universal donor cell (andcorresponding unmodified cell) may be a pluripotent stem cell (PSC). Insome embodiments, a cell, e.g., a universal donor cell (andcorresponding unmodified cell) may be an embryonic stem cell (ESC), anadult stem cell (ASC), an induced pluripotent stem cell (iPSC), or ahematopoietic stem or progenitor cell (HSPC). In some embodiments, acell, e.g., universal donor cell (and corresponding unmodified cell) maybe a differentiated cell. In some embodiments, a cell, e.g., universaldonor cell (and corresponding unmodified cell) may be a somatic cell,e.g., an immune system cell or a contractile cell, e.g., a skeletalmuscle cell.

The cells, e.g., universal donor stem cells, described herein may bedifferentiated into relevant cell types to assess HLA expression, aswell as the evaluation of immunogenicity of the universal stem celllines. In general, differentiation comprises maintaining the cells ofinterest for a period time and under conditions sufficient for the cellsto differentiate into the differentiated cells of interest. For example,the universal stem cells disclosed herein may be differentiated intomesenchymal progenitor cells (MPCs), hypoimmunogenic cardiomyocytes,muscle progenitor cells, blast cells, endothelial cells (ECs),macrophages, hepatocytes, beta cells (e.g., pancreatic beta cells),pancreatic endoderm progenitors, pancreatic endocrine progenitors, orneural progenitor cells (NPCs).

Stem cells are capable of both proliferation and giving rise to moreprogenitor cells, these in turn having the ability to generate a largenumber of mother cells that can in turn give rise to differentiated ordifferentiable daughter cells. The daughter cells themselves can beinduced to proliferate and produce progeny that subsequentlydifferentiate into one or more mature cell types, while also retainingone or more cells with parental developmental potential. The term “stemcell” refers then, to a cell with the capacity or potential, underparticular circumstances, to differentiate to a more specialized ordifferentiated phenotype, and which retains the capacity, under certaincircumstances, to proliferate without substantially differentiating. Inone aspect, the term progenitor or stem cell refers to a generalizedmother cell whose descendants (progeny) specialize, often in differentdirections, by differentiation, e.g., by acquiring completely individualcharacters, as occurs in progressive diversification of embryonic cellsand tissues. Cellular differentiation is a complex process typicallyoccurring through many cell divisions. A differentiated cell may derivefrom a multipotent cell that itself is derived from a multipotent cell,and so on. While each of these multipotent cells may be considered stemcells, the range of cell types that each can give rise to may varyconsiderably. Some differentiated cells also have the capacity to giverise to cells of greater developmental potential. Such capacity may benatural or may be induced artificially upon treatment with variousfactors. In many biological instances, stem cells can also be“multipotent” because they can produce progeny of more than one distinctcell type, but this is not required for “stem-ness.”

A “differentiated cell” is a cell that has progressed further down thedevelopmental pathway than the cell to which it is being compared. Thus,stem cells can differentiate into lineage-restricted precursor cells(such as a myocyte progenitor cell), which in turn can differentiateinto other types of precursor cells further down the pathway (such as amyocyte precursor), and then to an end-stage differentiated cell, suchas a myocyte, which plays a characteristic role in a certain tissuetype, and may or may not retain the capacity to proliferate further.

Embryonic Stem Cells

The cells described herein may be embryonic stem cells (ESCs). ESCs arederived from blastocytes of mammalian embryos and are able differentiateinto any cell type and propagate rapidly. ESCs are also believed to havea normal karyotype, maintaining high telomerase activity, and exhibitingremarkable long-term proliferative potential, making these cellsexcellent candidates for use as universal donor cells.

Adult Stem Cells

The cells described herein may be adult stem cells (ASCs). ASCs areundifferentiated cells that may be found in mammals, e.g., humans. ASCsare defined by their ability to self-renew, e.g., be passaged throughseveral rounds of cell replication while maintaining theirundifferentiated state, and ability to differentiate into severaldistinct cell types, e.g., glial cells. Adult stem cells are a broadclass of stem cells that may encompass hematopoietic stem cells, mammarystem cells, intestinal stem cells, mesenchymal stem cells, endothelialstem cells, neural stem cells, olfactory adult stem cells, neural creststem cells, and testicular cells.

Induced Pluripotent Stem Cells

The cells described herein may be induced pluripotent stem cells(iPSCs). An iPSC may be generated directly from an adult human cell byintroducing genes that encode critical transcription factors involved inpluripotency, e.g., Oct4, Sox2, cMyc, and Klf4. An iPSC may be derivedfrom the same subject to which subsequent progenitor cells are to beadministered. That is, a somatic cell can be obtained from a subject,reprogrammed to an induced pluripotent stem cell, and thenre-differentiated into a progenitor cell to be administered to thesubject (e.g., autologous cells). However, in the case of autologouscells, a risk of immune response and poor viability post-engraftmentremain.

Human Hematopoietic Stem and Progenitor Cells

The cells described herein may be human hematopoietic stem andprogenitor cells (hHSPCs). This stem cell lineage gives rise to allblood cell types, including erythroid (erythrocytes or red blood cells(RBCs)), myeloid (monocytes and macrophages, neutrophils, basophils,eosinophils, megakaryocytes/platelets, and dendritic cells), andlymphoid (T-cells, B-cells, NK-cells). Blood cells are produced by theproliferation and differentiation of a very small population ofpluripotent hematopoietic stem cells (HSCs) that also have the abilityto replenish themselves by self-renewal. During differentiation, theprogeny of HSCs progress through various intermediate maturationalstages, generating multi-potential and lineage-committed progenitorcells prior to reaching maturity. Bone marrow (BM) is the major site ofhematopoiesis in humans and, under normal conditions, only small numbersof hematopoietic stem and progenitor cells (HSPCs) can be found in theperipheral blood (PB). Treatment with cytokines, some myelosuppressivedrugs used in cancer treatment, and compounds that disrupt theinteraction between hematopoietic and BM stromal cells can rapidlymobilize large numbers of stem and progenitors into the circulation.

Differentiation of Cells into Other Cell Types

Another step of the methods of the present disclosure may comprisedifferentiating cells into differentiated cells. The differentiatingstep may be performed according to any method known in the art. Forexample, human iPSCs are differentiated into definitive endoderm usingvarious treatments, including activin and B27 supplement (LifeTechnologies). The definitive endoderm is further differentiated intohepatocyte, the treatment includes: FGF4, HGF, BMP2, BMP4, Oncostatin M,Dexametason, etc. (Duan et al, Stem Cells, 2010; 28:674-686; Ma et al,Stem Cells Translational Medicine, 2013; 2:409-419). In anotherembodiment, the differentiating step may be performed according toSawitza et al, Sci Rep. 2015; 5: 13320. A differentiated cell may be anysomatic cell of a mammal, e.g., a human. In some embodiments, a somaticcell may be an endocrine secretory epithelial cell (e.g., thyroidhormone secreting cells, adrenal cortical cells), an exocrine secretoryepithelial cell (e.g., salivary gland mucous cell, prostate gland cell),a hormone-secreting cell (e.g., anterior pituitary cell, pancreaticislet cell), a keratinizing epithelial cell (e.g., epidermalkeratinocyte), a wet stratified barrier epithelial cell, a sensorytransducer cell (e.g., a photoreceptor), an autonomic neuron cells, asense organ and peripheral neuron supporting cell (e.g., Schwann cell),a central nervous system neuron, a glial cell (e.g., astrocyte,oligodendrocyte), a lens cell, an adipocyte, a kidney cell, a barrierfunction cell (e.g., a duct cell), an extracellular matrix cell, acontractile cell (e.g., skeletal muscle cell, heart muscle cell, smoothmuscle cell), a blood cell (e.g., erythrocyte), an immune system cell(e.g., megakaryocyte, microglial cell, neutrophil, mast cell, a T cell,a B cell, a Natural Killer cell), a germ cell (e.g., spermatid), a nursecell, or an interstitial cell.

V. Formulations and Administrations

Formulation and Delivery for Gene Editing

Guide RNAs, polynucleotides, e.g., polynucleotides that encode atolerogenic factor or polynucleotides that encode an endonuclease, andendonucleases as described herein may be formulated and delivered tocells in any manner known in the art.

Guide RNAs and/or polynucleotides may be formulated withpharmaceutically acceptable excipients such as carriers, solvents,stabilizers, adjuvants, diluents, etc., depending upon the particularmode of administration and dosage form. Guide RNAs and/orpolynucleotides compositions can be formulated to achieve aphysiologically compatible pH, and range from a pH of about 3 to a pH ofabout 11, about pH 3 to about pH 7, depending on the formulation androute of administration. In some cases, the pH can be adjusted to arange from about pH 5.0 to about pH 8. In some cases, the compositionscan comprise a therapeutically effective amount of at least one compoundas described herein, together with one or more pharmaceuticallyacceptable excipients. Optionally, the compositions can comprise acombination of the compounds described herein, or can include a secondactive ingredient useful in the treatment or prevention of bacterialgrowth (for example and without limitation, anti-bacterial oranti-microbial agents), or can include a combination of reagents of thepresent disclosure.

Suitable excipients include, for example, carrier molecules that includelarge, slowly metabolized macromolecules such as proteins,polysaccharides, polylactic acids, polyglycolic acids, polymeric aminoacids, amino acid copolymers, and inactive virus particles. Otherexemplary excipients can include antioxidants (for example and withoutlimitation, ascorbic acid), chelating agents (for example and withoutlimitation, EDTA), carbohydrates (for example and without limitation,dextrin, hydroxyalkylcellulose, and hydroxyalkylmethylcellulose),stearic acid, liquids (for example and without limitation, oils, water,saline, glycerol and ethanol), wetting or emulsifying agents, pHbuffering substances, and the like.

Guide RNA polynucleotides (RNA or DNA) and/or endonucleasepolynucleotide(s) (RNA or DNA) can be delivered by viral or non-viraldelivery vehicles known in the art. Alternatively, endonucleasepolypeptide(s) can be delivered by viral or non-viral delivery vehiclesknown in the art, such as electroporation or lipid nanoparticles. Infurther alternative aspects, the DNA endonuclease can be delivered asone or more polypeptides, either alone or pre-complexed with one or moreguide RNAs, or one or more crRNA together with a tracrRNA.

Polynucleotides can be delivered by non-viral delivery vehiclesincluding, but not limited to, nanoparticles, liposomes,ribonucleoproteins, positively charged peptides, small moleculeRNA-conjugates, aptamer-RNA chimeras, and RNA-fusion protein complexes.Some exemplary non-viral delivery vehicles are described in Peer andLieberman, Gene Therapy, 2011, 18: 1127-1133 (which focuses on non-viraldelivery vehicles for siRNA that are also useful for delivery of otherpolynucleotides).

For polynucleotides of the disclosure, the formulation may be selectedfrom any of those taught, for example, in International ApplicationPCT/US2012/069610.

Polynucleotides, such as guide RNA, sgRNA, and mRNA encoding anendonuclease, may be delivered to a cell or a subject by a lipidnanoparticle (LNP).

A LNP refers to any particle having a diameter of less than 1000 nm, 500nm, 250 nm, 200 nm, 150 nm, 100 nm, 75 nm, 50 nm, or 25 nm.Alternatively, a nanoparticle may range in size from 1-1000 nm, 1-500nm, 1-250 nm, 25-200 nm, 25-100 nm, 35-75 nm, or 25-60 nm.

LNPs may be made from cationic, anionic, or neutral lipids. Neutrallipids, such as the fusogenic phospholipid DOPE or the membranecomponent cholesterol, may be included in LNPs as ‘helper lipids’ toenhance transfection activity and nanoparticle stability. Limitations ofcationic lipids include low efficacy owing to poor stability and rapidclearance, as well as the generation of inflammatory oranti-inflammatory responses.

LNPs may also be comprised of hydrophobic lipids, hydrophilic lipids, orboth hydrophobic and hydrophilic lipids.

Any lipid or combination of lipids that are known in the art can be usedto produce a LNP. Examples of lipids used to produce LNPs are: DOTMA,DOSPA, DOTAP, DMRIE, DC-cholesterol, DOTAP-cholesterol,GAP-DMORIE-DPyPE, and GL67A-DOPE-DMPE-polyethylene glycol (PEG).Examples of cationic lipids are: 98N12-5, C12-200, DLin-KC2-DMA (KC2),DLin-MC3-DMA (MC3), XTC, MD1, and 7C1. Examples of neutral lipids are:DPSC, DPPC, POPC, DOPE, and SM. Examples of PEG-modified lipids are:PEG-DMG, PEG-CerC14, and PEG-CerC20.

The lipids can be combined in any number of molar ratios to produce aLNP. In addition, the polynucleotide(s) can be combined with lipid(s) ina wide range of molar ratios to produce a LNP.

A recombinant adeno-associated virus (AAV) vector can be used fordelivery. Techniques to produce rAAV particles, in which an AAV genometo be packaged that includes the polynucleotide to be delivered, rep andcap genes, and helper virus functions are provided to a cell arestandard in the art. Production of rAAV typically requires that thefollowing components are present within a single cell (denoted herein asa packaging cell): a rAAV genome, AAV rep and cap genes separate from(i.e., not in) the rAAV genome, and helper virus functions. The AAV repand cap genes may be from any AAV serotype for which recombinant viruscan be derived, and may be from a different AAV serotype than the rAAVgenome ITRs, including, but not limited to, AAV serotypes describedherein. Production of pseudotyped rAAV is disclosed in, for example,international patent application publication number WO 01/83692.

Formulation and Administration of Cells, e.g., Universal Donor Cells

Genetically modified cells, e.g., universal donor cells, as describedherein may be formulated and administered to a subject by any mannerknown in the art.

The terms “administering,” “introducing”, “implanting”, “engrafting” and“transplanting” are used interchangeably in the context of the placementof cells, e.g., progenitor cells, into a subject, by a method or routethat results in at least partial localization of the introduced cells ata desired site. The cells e.g., progenitor cells, or theirdifferentiated progeny can be administered by any appropriate route thatresults in delivery to a desired location in the subject where at leasta portion of the implanted cells or components of the cells remainviable. The period of viability of the cells after administration to asubject can be as short as a few hours, e.g., twenty-four hours, to afew days, to as long as several years, or even the life time of thesubject, i.e., long-term engraftment.

A genetically modified cell, e.g., universal donor cell, as describedherein may be viable after administration to a subject for a period thatis longer than that of an unmodified cell.

In some embodiments, a composition comprising cells as described hereinmay be administered by a suitable route, which may include intravenousadministration, e.g., as a bolus or by continuous infusion over a periodof time. In some embodiments, intravenous administration may beperformed by intramuscular, intraperitoneal, intracerebrospinal,subcutaneous, intra-articular, intrasynovial, or intrathecal routes. Insome embodiments, a composition may be in solid form, aqueous form, or aliquid form. In some embodiments, an aqueous or liquid form may benebulized or lyophilized. In some embodiments, a nebulized orlyophilized form may be reconstituted with an aqueous or liquidsolution.

A cell composition can also be emulsified or presented as a liposomecomposition, provided that the emulsification procedure does notadversely affect cell viability. The cells and any other activeingredient can be mixed with excipients that are pharmaceuticallyacceptable and compatible with the active ingredient, and in amountssuitable for use in the therapeutic methods described herein.

Additional agents included in a cell composition can includepharmaceutically acceptable salts of the components therein.Pharmaceutically acceptable salts include the acid addition salts(formed with the free amino groups of the polypeptide) that are formedwith inorganic acids, such as, for example, hydrochloric or phosphoricacids, or such organic acids as acetic, tartaric, mandelic and the like.Salts formed with the free carboxyl groups can also be derived frominorganic bases, such as, for example, sodium, potassium, ammonium,calcium or ferric hydroxides, and such organic bases as isopropylamine,trimethylamine, 2-ethylamino ethanol, histidine, procaine and the like.

Physiologically tolerable carriers are well known in the art. Exemplaryliquid carriers are sterile aqueous solutions that contain no materialsin addition to the active ingredients and water, or contain a buffersuch as sodium phosphate at physiological pH value, physiological salineor both, such as phosphate-buffered saline. Still further, aqueouscarriers can contain more than one buffer salt, as well as salts such assodium and potassium chlorides, dextrose, polyethylene glycol and othersolutes. Liquid compositions can also contain liquid phases in additionto and to the exclusion of water. Exemplary of such additional liquidphases are glycerin, vegetable oils such as cottonseed oil, andwater-oil emulsions. The amount of an active compound used in the cellcompositions that is effective in the treatment of a particular disorderor condition can depend on the nature of the disorder or condition, andcan be determined by standard clinical techniques.

In some embodiments, a composition comprising cells may be administeredto a subject, e.g., a human subject, who has, is suspected of having, oris at risk for a disease. In some embodiments, a composition may beadministered to a subject who does not have, is not suspected of havingor is not at risk for a disease. In some embodiments, a subject is ahealthy human. In some embodiments, a subject e.g., a human subject, whohas, is suspected of having, or is at risk for a genetically inheritabledisease. In some embodiments, the subject is suffering or is at risk ofdeveloping symptoms indicative of a disease.

VI. Specific Compositions and Methods of the Disclosure

Accordingly, the present disclosure relates in particular to thefollowing non-limiting compositions and methods.

In a first composition, Composition 1, the present disclosure acomposition comprising cells that comprise (i) at least one geneticmodification within or near at least one gene that encodes one or moreMHC-I and MHC-II human leukocyte antigens or other components ortranscriptional regulators of the MHC-I or MHC-II complex; (ii) at leastone genetic modification that increases the expression of at least onepolynucleotide that encodes a tolerogenic factor relative to anunmodified cell; and (iii) at least one genetic modification thatincreases or decreases the expression of at least one gene that encodesa survival factor relative to an unmodified cell.

In another composition, Composition 2, the present disclosure provides acomposition comprising cells that comprise (i) at least one deletionand/or insertion of at least one base pair within or near at least onegene that encodes one or more MHC-I and MHC-II human leukocyte antigensor other components or transcriptional regulators of the MHC-I or MHC-IIcomplex; and (ii) at least one insertion of a polynucleotide thatencodes at least one tolerogenic factor at a site that partiallyoverlaps, completely overlaps, or is contained within, the site of agenetic deletion of (i).

In another composition, Composition 3, the present disclosure provides acomposition comprising cells that comprise at least one geneticmodification that increases or decreases the expression of at least onegene that encodes a survival factor relative to an unmodified cell.

In another composition, Composition 4, the present disclosure provides acomposition, as provided in Composition 1, wherein the geneticmodification of (i) is a deletion.

In another composition, Composition 5, the present disclosure provides acomposition, as provided in Composition 1, wherein the geneticmodification of (ii) is an insertion of a polynucleotide that encodes atolerogenic factor at a safe harbor locus or at a site that partiallyoverlaps, completely overlaps, or is contained within, the site of agenetic modification of (i).

In another composition, Composition 6, the present disclosure provides acomposition, as provided in Composition 1, wherein the geneticmodification of (i) is a deletion of a gene that encodes one or moreMHC-I and MHC-II human leukocyte antigens or other components ortranscriptional regulators of the MHC-I or MHC-II complex; and thegenetic modification of (ii) is an insertion of a polynucleotide thatencodes at least one tolerogenic factor at a site that partiallyoverlaps, completely overlaps, or is contained within, the site of agenetic modification of (i).

In another composition, Composition 7, the present disclosure provides acomposition, as provided in any one of Compositions 1, 2, or 4 to 6,wherein the at least one gene that encodes one or more MHC-I and MHC-IIhuman leukocyte antigens or other components or transcriptionalregulators of the MHC-I or MHC-II complex is one or more of a MHC-I gene(e.g., HLA-A, HLA-B and HLA-C), a MHC-II gene (e.g., HLA-DP, HLA-DM,HLA-DOA, HLA-DOB, HLA-DQ, and HLA-DR), or a gene that encodes atranscriptional regulator of MHC-I or MHC-II or other component of theMHC-I complex (e.g., B2M, NLRC5, and CIITA).

In another composition, Composition 8, the present disclosure provides acomposition, as provided in of any one of Compositions 1, 2, or 4 to 6,wherein the at least one gene that encodes one or more MHC-I and MHC-IIhuman leukocyte antigens or other components or transcriptionalregulators of the MHC-I or MHC-II complex is one or more of HLA-A,HLA-B, HLA-C, B2M, or CIITA.

In another composition, Composition 9, the present disclosure provides acomposition, as provided in Composition 1 or 2, wherein (i) is adeletion within or near one or more of HLA-A, HLA-B, HLA-C, B2M, orCIITA.

In another composition, Composition 10, the present disclosure providesa composition, as provided in Composition 9, wherein (i) is a deletionwithin or near HLA-A, a deletion within or near HLA-B, a deletion withinor near HLA-C, or a deletion within or near B2M.

In another composition, Composition 11, the present disclosure providesa composition, as provided in Composition 9, wherein (i) is a deletionwithin or near HLA-A, a deletion within or near HLA-B, a deletion withinor near HLA-C, or a deletion within or near CIITA.

In another composition, Composition 12, the present disclosure providesa composition, as provided in any one of Compositions 1, 2, or 4 to 11,wherein the at least one polynucleotide that encodes a tolerogenicfactor is one or more polynucleotides that encode one or more of HLA-E,HLA-G, CTLA-4, CD47, or PD-L1.

In another composition, Composition 13, the present disclosure providesa composition, as provided in Composition 12, wherein (i) is a deletionwithin or near B2M and (ii) is an insertion of a polynucleotide encodingPD-L1 at a site that partially overlaps, completely overlaps, or iscontained within the deletion in (i).

In another composition, Composition 14, the present disclosure providesa composition, as provided in Composition 12, wherein (i) is a deletionwithin or near HLA-A, a deletion within or near HLA-B, or a deletionwithin or near HLA-C and (ii) is an insertion of a polynucleotide thatencodes HLA-G at a site that partially overlaps, completely overlaps, oris contained within a deletion in (i) (e.g., the HLA-A deletion).

In another composition, Composition 15, the present disclosure providesa composition, as provided in Composition 12, wherein (i) is a deletionwithin or near HLA-A, a deletion within or near HLA-B, a deletion withinor near HLA-C, or a deletion within or near CIITA and (ii) is aninsertion of a polynucleotide that encodes HLA-G at a site thatpartially overlaps, completely overlaps, or is contained within thedeletion within or near HLA-A and insertion of a polynucleotide thatencodes CD47 at a site that partially overlaps, completely overlaps, oris contained within the deletion within or near CIITA.

In another composition, Composition 16, the present disclosure providesa composition, as provided in any one of Compositions 1 or 3 to 15,wherein the at least one gene that encodes a survival factor is one ormore genes that encode one or more of ZNF143, TXNIP, FOXO1, JNK, orMANF.

In another composition, Composition 17, the present disclosure providesa composition, as provided in any one of Compositions 1 or 3 to 16,wherein the genetic modification that increases or decreases theexpression of a gene that encodes a survival factor relative to anunmodified cell is an insertion of a polynucleotide that encodes MANF,e.g., at a safe harbor locus.

In another composition, Composition 18, the present disclosure providesa composition, as provided in any one of Compositions 1 or 3 to 16,wherein the genetic modification that increases or decreases theexpression of a gene that encodes a survival factor relative to anunmodified cell is a deletion within or near a ZNF143, TXNIP, FOXO1, orINK gene, which reduces or eliminates expression of the ZNF143, TXNIP,FOXO1, or JNK gene relative to an unmodified cell.

In another composition, Composition 19, the present disclosure providesa composition, as provided in any one of Compositions 1 to 18, whereinthe cells further comprise an exogenous polynucleotide that is notintegrated into the genomic DNA of the cells.

In another composition, Composition 20, the present disclosure providesa composition, as provided in Composition 19, wherein the exogenouspolynucleotide encodes HLA-E, HLA-G, CTLA-4, CD47, MANF, and/or PD-L1.

In another composition, Composition 21, the present disclosure providesa composition, as provided in any one of Compositions 1 to 20, whereinthe cells further comprise increased expression of one or more safetyswitch relative to an unmodified cell.

In another composition, Composition 22, the present disclosure providesa composition, as provided in Composition 21, wherein a safety switch isherpes simplex virus-1 thymidine kinase (HSV-tk) or inducible caspase-9.

In another composition, Composition 23, the present disclosure providesa composition, as provided in Composition 21 or 22, wherein increasedexpression of one or more safety switches results from a geneticinsertion of a polynucleotide that encodes a safety switch protein,e.g., into a safe harbor locus.

In another composition, Composition 24, the present disclosure providesa composition, as provided in any one of Compositions 5, 17, or 23,wherein the safe harbor locus is selected from the group consisting ofAAVS1 (PPP1 R12C), ALB, Angptl3, ApoC3, ASGR2, CCR5, FIX (F9), G6PC,Gys2, HGD, Lp(a), Pcsk9, Serpinal, TF, and TTR.

In another composition, Composition 25, the present disclosure providesa composition, as provided in any one of Compositions 1 to 24, whereinthe cells further comprise an additional genetic modification thatdecreases the expression of any additional gene.

In another composition, Composition 26, the present disclosure providesa composition, as provided in any one of Compositions 1 to 25, whereinthe genetic modification, genetic deletion, or genetic insertion isproduced by delivering to the cells an endonuclease and a guide RNA(gRNA).

In another composition, Composition 27, the present disclosure providesa composition, as provided in Composition 26, wherein the endonucleaseis a Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cash, Cas7, Cas8, Cas9 (alsoknown as Csn1 and Csx12), Cas100, Csy1, Csy2, Csy3, Cse1, Cse2, Csc1,Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5,Cmr6, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csx1,Csx15, Csf1, Csf2, Csf3, Csf4, or Cpf1 endonuclease; a homolog thereof,a recombination of the naturally occurring molecule thereof,codon-optimized thereof, or modified versions thereof, and combinationsthereof.

In another composition, Composition 28, the present disclosure providesa composition, as provided in Composition 27, wherein the endonucleaseis a Cas9, optionally a S. pyogenes Cas9, or a variant thereofcomprising a N-terminus SV40 NLS and a C-terminus SV40 NLS.

In another composition, Composition 29, the present disclosure providesa composition, as provided in Composition 26, wherein the weight ratioof said gRNA to said endonuclease is 1:1.

In another composition, Composition 30, the present disclosure providesa composition, as provided in any one of Compositions 2, 5, 6, 12 to 15,17, 19, 20, or 23 wherein the polynucleotide comprises an exogenouspromoter.

In another composition, Composition 31, the present disclosure providesa composition, as provided in Composition 30, wherein the exogenouspromoter is a CMV, EF1a, PGK, CAG, UBC, or other constitutive,inducible, temporal-, tissue-, or cell type-specific promoter.

In another composition, Composition 32, the present disclosure providesa composition, as provided in Composition 31, wherein the exogenouspromoter is a CAG promoter.

In another composition, Composition 33, the present disclosure providesa composition, as provided in any one of Compositions 1 to 32, whereinthe cells are stem cells (e.g., human stem cells).

In another composition, Composition 34, the present disclosure providesa composition, as provided in any one of Compositions 1 to 33, whereinthe cells are embryonic stem cells (ESCs), adult stem cells (ASCs),induced pluripotent stem cells (iPSCs), or hematopoietic stem andprogenitor cells (HSPCs).

In another composition, Composition 35, the present disclosure providesa composition, as provided in any one of Compositions 1 to 32, whereinthe cells are differentiated cells.

In another composition, Composition 36, the present disclosure providesa composition, as provided in any one of Composition 1 to 32 or 35,wherein the cells are somatic cells.

In a first method, Method 1, the present disclosure provides a method ofgenerating modified cells, the method comprising: (i) introducing atleast one genetic modification within or near at least one gene thatencodes one or more MHC-I and MHC-II human leukocyte antigens or othercomponents or transcriptional regulators of the MHC-I or MHC-II complex;(ii) introducing at least one genetic modification that increases theexpression of at least one polynucleotide that encodes a tolerogenicfactor in the cells; and (iii) introducing at least one geneticmodification that increases or decreases the expression of at least onegene that encodes a survival factor in the universal donor cells.

In another method, Method 2, the present disclosure provides a method ofgenerating universal donor cells, the method comprising: (i) introducingat least one deletion of at least one region of genomic DNA within ornear at least one gene that encodes one or more MHC-I and MHC-II humanleukocyte antigens or other components or transcriptional regulators ofthe MHC-I or MHC-II complex; and (ii) introducing at least one insertionof at least one polynucleotide that encodes a tolerogenic factor at asite that partially overlaps, completely overlaps, or is containedwithin, the site of a deletion of (i).

In another method, Method 3, the present disclosure provides a method ofgenerating universal donor cells, the method comprising introducing atleast one genetic modification that increases or decreases theexpression of at least one gene that encodes a survival factor.

In another method, Method 4, the present disclosure provides a method asprovided in Method 1, wherein the genetic modification of (i) is adeletion.

In another method, Method 5, the present disclosure provides a method asprovided in Method 1, wherein the genetic modification of (ii) is aninsertion of a polynucleotide that encodes a tolerogenic factor at asafe harbor locus or at a site that partially overlaps, completelyoverlaps, or is contained within, the site of a genetic modification of(i).

In another method, Method 6, the present disclosure provides a method asprovided in Method 1, wherein the genetic modification of (i) is adeletion of a gene that encodes one or more MHC-I and MHC-II humanleukocyte antigens or other components or transcriptional regulators ofthe MHC-I or MHC-II complex; and the genetic modification of (ii) is aninsertion of a polynucleotide that encodes at least one tolerogenicfactor at a site that partially overlaps, completely overlaps, or iscontained within, the site of a genetic modification of (i).

In another method, Method 7, the present disclosure provides a method asprovided in any one of Methods 1, 2, or 4 to 6, wherein the at least onegene that encodes one or more MHC-I and MHC-II human leukocyte antigensor other components or transcriptional regulators of the MHC-I or MHC-IIcomplex is one or more of a MHC-I gene (e.g., HLA-A, HLA-B and HLA-C), aMHC-II gene (e.g., HLA-DP, HLA-DM, HLA-DOA, HLA-DOB, HLA-DQ, andHLA-DR), or a gene that encodes a transcriptional regulator of MHC-I orMHC-II (e.g., B2M, NLRC5, and CIITA).

In another method, Method 8, the present disclosure provides a method asprovided in any one of Methods 1, 2, or 4 to 6, wherein the at least onegene that encodes one or more MHC-I and MHC-II human leukocyte antigensor other components or transcriptional regulators of the MHC-I or MHC-IIcomplex is one or more of HLA-A, HLA-B, HLA-C, B2M, or CIITA.

In another method, Method 9, the present disclosure provides a method asprovided in Method 1 or 2, wherein (i) is a deletion within or near oneor more of HLA-A, HLA-B, HLA-C, B2M, or CIITA.

In another method, Method 10, the present disclosure provides a methodas provided in Method 9, wherein (i) is a deletion within or near HLA-A,a deletion within or near HLA-B, a deletion within or near HLA-C, and adeletion within or near B2M.

In another method, Method 11, the present disclosure provides a methodas provided in Method 9, wherein (i) is a deletion within or near HLA-A,a deletion within or near HLA-B, a deletion within or near HLA-C, and adeletion within or near CIITA.

In another method, Method 12, the present disclosure provides a methodas provided in any one of Methods 1, 2, or 4 to 11, wherein the at leastone polynucleotide that encodes a tolerogenic factor is one or morepolynucleotides that encode one or more of HLA-E, HLA-G, CTLA-4, CD47,or PD-L1.

In another method, Method 13, the present disclosure provides a methodas provided in Method 12, wherein (i) is a deletion within or near B2Mand (ii) is an insertion of a polynucleotide encoding PD-L1 at a sitethat partially overlaps, completely overlaps, or is contained within thedeletion in (i).

In another method, Method 14, the present disclosure provides a methodas provided in Method 12, wherein (i) is a deletion within or nearHLA-A, a deletion within or near HLA-B, and a deletion within or nearHLA-C and (ii) is an insertion of a polynucleotide that encodes HLA-G ata site that partially overlaps, completely overlaps, or is containedwithin a deletion in (i) (e.g., the HLA-A deletion).

In another method, Method 15, the present disclosure provides a methodas provided in Method 12, wherein (i) is a deletion within or nearHLA-A, a deletion within or near HLA-B, a deletion within or near HLA-C,and a deletion within or near CIITA and (ii) is an insertion of apolynucleotide that encodes HLA-G at a site that partially overlaps,completely overlaps, or is contained within the deletion within or nearHLA-A and insertion of a polynucleotide that encodes CD47 at a site thatpartially overlaps, completely overlaps, or is contained within thedeletion within or near CIITA.

In another method, Method 16, the present disclosure provides a methodas provided in any one of Methods 1 or 3 to 15, wherein the at least onegene that encodes a survival factor is one or more genes that encode oneor more of a ZNF143, TXNIP, FOXO1, JNK, or MANF.

In another method, Method 17, the present disclosure provides a methodas provided in any one of Methods 1 or 3 to 16, wherein the geneticmodification that increases or decreases the expression of a gene thatencodes a survival factor is an insertion of a polynucleotide thatencodes MANF, e.g., at a safe harbor locus.

In another method, Method 18, the present disclosure provides a methodas provided in any one of Methods 1 or 3 to 16, wherein the geneticmodification that increases or decreases the expression of a gene thatencodes a survival factor is a deletion within or near a ZNF143, TXNIP,FOXO1, or JNK gene, which reduces or eliminates expression of theZNF143, TXNIP, FOXO1, or JNK gene relative to an unmodified cell.

In another method, Method 19, the present disclosure provides a methodas provided in any one of Methods 1 to 18, wherein the cells are stemcells (e.g., human stem cells).

In another method, Method 20, the present disclosure provides a methodas provided in any one of Methods 1 to 19, wherein the cells areembryonic stem cells (ESCs), adult stem cells (ASCs), inducedpluripotent stem cells (iPSCs), or hematopoietic stem and progenitorcells (HSPCs).

In another method, Method 21, the present disclosure provides a methodas provided in any one of Methods 1 to 18, wherein the cells aredifferentiated cells.

In another method, Method 22, the present disclosure provides a methodas provided in any one of Methods 1 to 18 or 21, wherein the cells aresomatic cells.

In another method, Method 23, the present disclosure provides a methodas provided in any one of Methods 1 to 22, wherein the method furthercomprises introducing an exogenous polynucleotide into the cells thatdoes not become integrated into the genomic DNA of the cells.

In another method, Method 24, the present disclosure provides a methodas provided in Method 23, wherein the exogenous polynucleotide encodesHLA-E, HLA-G, CTLA-4, CD47, MANF, and/or PD-L1.

In another method, Method 25, the present disclosure provides a methodas provided in any one of Methods 1 to 24, wherein the method furthercomprises increasing expression of one or more safety switches relativeto an unmodified cell.

In another method, Method 26, the present disclosure provides a methodas provided in Method 25, wherein a safety switch is herpes simplexvirus-1 thymidine kinase (HSV-tk) or inducible caspase-9.

In another method, Method 27, the present disclosure provides a methodas provided in Method 25 or 26, wherein increasing expression of one ormore safety switches results from a genetic insertion of apolynucleotide that encodes a safety switch, e.g., into a safe harborlocus.

In another method, Method 28, the present disclosure provides a methodas provided in any one of Methods 5, 17, or 27, wherein the safe harborlocus is selected from the group consisting of AAVS1 (PPP1 R12C), ALB,Angptl3, ApoC3, ASGR2, CCR5, FIX (F9), G6PC, Gys2, HGD, Lp(a), Pcsk9,Serpinal, TF, and TTR.

In another method, Method 29, the present disclosure provides a methodas provided in any one of Methods 1 to 28, wherein the method furthercomprises introducing an additional genetic modification that decreasesthe expression of any additional gene.

In another method, Method 30, the present disclosure provides a methodas provided in any one of Methods 1 to 29, wherein the geneticmodification, deletion, or insertion is produced by delivering to thecells an endonuclease and at least one guide RNA (gRNA).

In another method, Method 31, the present disclosure provides a methodas provided in Method 30, wherein the endonuclease is a Cas1, Cas1B,Cas2, Cas3, Cas4, Cas5, Cash, Cas7, Cas8, Cas9 (also known as Csn1 andCsx12), Cas100, Csy1, Csy2, Csy3, Cse1, Cse2, Csc1, Csc2, Csa5, Csn2,Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2,Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csx1, Csx15, Csf1, Csf2,Csf3, Csf4, or Cpf1 endonuclease; a homolog thereof, a recombination ofthe naturally occurring molecule thereof, codon-optimized thereof, ormodified versions thereof, and combinations thereof.

In another method, Method 32, the present disclosure provides a methodas provided in Method 31, wherein the endonuclease is a Cas9, optionallya S. pyogenes Cas9, or a variant thereof comprising an N-terminus SV40NLS and a C-terminus SV40 NLS.

In another method, Method 33, the present disclosure provides a methodas provided in Method 30, wherein the weight ratio of said gRNA(s) tosaid endonuclease is 1:1.

In another method, Method 34, the present disclosure provides a methodas provided in any one of Methods 2, 5, 6, 12 to 15, 17, 23, 25, or 27,wherein the polynucleotide comprises an exogenous promoter.

In another method, Method 35, the present disclosure provides a methodas provided in Method 34, wherein the exogenous promoter is a CMV, EF1a,PGK, CAG, UBC, or other constitutive, inducible, temporal-, tissue-, orcell type-specific promoter.

In another method, Method 36, the present disclosure provides a methodas provided in Method 35, wherein the exogenous promoter is a CAGpromoter.

In another method, Method 37, the present disclosure provides a methodcomprising administering to a subject a composition of cells as providedin any one of Compositions 1 to 36 or a composition comprising aplurality of cells generated by any one of Methods 1 to 36.

In another method, Method 38, the present disclosure provides a methodcomprising (i) obtaining a composition of cells as provided in any oneof Compositions 1 to 34; (ii) differentiating the cells intolineage-restricted cells or fully differentiated cells; and (iii)administering the lineage-restricted cells or fully differentiated cellsto a subject in need thereof.

In another method, Method 39, the present disclosure provides a methodas provided in Method 37 or 38, wherein the subject is a human who has,is suspected of having, or is at risk for a disease.

In another method, Method 40, the present disclosure provides a methodas provided in Method 39, wherein the disease is a geneticallyinheritable disease.

In another method, Method 41, the present disclosure provides a methodas provided in Method 39 or 40, wherein the cells further comprise agenetic modification that decreases the expression of a gene or proteinthat is associated with the disease.

In another method, Method 42, the present disclosure provides a methodas provided in any one of Methods 39 to 41, wherein the geneticmodification is capable of treating the disease or symptoms of thedisease.

In another method, Method 43, the present disclosure provides a methodas provided in any one of Methods 37 to 42, wherein the cells areobtained from a source other than the subject.

In another method, Method 44, the present disclosure provides a methodof generating a universal donor cell, the method comprising geneticallymodifying a cell by (i) introducing a deletion and/or insertion of atleast one base pair in the genome of the cell at a site within or nearat least one gene that encodes one or more of a MHC-I or MHC-II humanleukocyte antigens or a component or a transcriptional regulator of aMHC-I or MHC-II complex; and (ii) introducing in the genome of the cellan insertion of at least one polynucleotide that encodes a tolerogenicfactor, at a site that partially overlaps, completely overlaps, or iscontained within, the site of (i), thereby generating the universaldonor cell.

In another method, Method 45, the present disclosure provides a methodof generating a universal donor cell, the method comprising geneticallymodifying a cell by (i) introducing a deletion and/or insertion of atleast one base pair in the genome of the cell at a site within or nearat least one gene that encodes one or more of a MHC-I or MHC-II humanleukocyte antigens or a component or a transcriptional regulator of aMHC-I or MHC-II complex; and (ii) introducing in the genome of the cellan insertion of at least one polynucleotide that encodes a tolerogenicfactor into a safe harbor locus, thereby generating the universal donorcell.

In another method, Method 46, the present disclosure provides a methodas provided in Methods 44 or 45, wherein the universal donor cell hasincreased immune evasion and/or cell survival compared to an unmodifiedcell.

In another method, Method 47, the present disclosure provides a methodas provided in any one of Methods 44 to 46, wherein the at least onegene that encodes one or more MHC-I or MHC-II human leukocyte antigensor the component or the transcriptional regulator of the MHC-I or MHC-IIcomplex is a MHC-I gene chosen from HLA-A, HLA-B, or HLA-C, a MHC-IIgene chosen from HLA-DP, HLA-DM, HLA-DOA, HLA-DOB, HLA-DQ, or HLA-DR, ora gene chosen from B2M, NLRC5, CIITA, RFX5, RFXAP, or RFXANK.

In another method, Method 48, the present disclosure provides a methodas provided in any one of Methods 44 to 47, wherein the at least onepolynucleotide that encodes a tolerogenic factor is one or morepolynucleotides that encode one or more of PD-L1, HLA-E, HLA-G, CTLA-4,or CD47.

In another method, Method 49, the present disclosure provides a methodas provided in any one of Methods 44 to 48, wherein the at least onepolynucleotide that encodes a tolerogenic factor is operably linked toan exogenous promoter.

In another method, Method 50, the present disclosure provides a methodas provided in Method 49, wherein the exogenous promoter is aconstitutive, inducible, temporal-, tissue-, or cell type-specificpromoter, the constitutive promoter being a CMV, EF1a, PGK, CAG, or UBCpromoter.

In another method, Method 51, the present disclosure provides a methodas provided in any one of Methods 44 to 50, wherein the deletion and/orinsertion of (i) is within or near B2M, and the insertion of (ii) is apolynucleotide encoding PD-L1 or HLA-E.

In another method, Method 52, the present disclosure provides a methodas provided in any one of Methods 44 to 51, wherein the method furthercomprises introducing at least one genetic modification that increasesor decreases expression of at least a survival factor relative to anunmodified cell.

In another method, Method 53, the present disclosure provides a methodas provided in Method 52, wherein the at least one genetic modificationthat increases or decreases expression of at least a survival factor isan insertion of a polynucleotide that encodes MANF, which increasesexpression of MANF relative to the unmodified cell; or a deletion and/orinsertion of at least one base pair within or near a gene that encodesZNF143, TXNIP, FOXO1, or JNK, which reduces or eliminates expression ofZNF143, TXNIP, FOXO1, or JNK relative to the unmodified cell.

In another method, Method 54, the present disclosure provides a methodas provided in Method 53, wherein the polynucleotide that encodes MANFis inserted into a safe harbor locus or into a gene belonging to aMHC-I, MHC-II, or transcriptional regulator of MHC-I or MHC-II.

In another method, Method 55, the present disclosure provides a methodas provided in any one of Methods 44 to 54, wherein the geneticallymodifying comprises delivering at least one RNA-guided endonucleasesystem to the cell.

In another method, Method 56, the present disclosure provides a methodas provided in Method 55, wherein the at least one RNA-guidedendonuclease system is a CRISPR system comprising a CRISPR nuclease anda guide RNA.

In another method, Method 57, the present disclosure provides a methodas provided in Method 56, wherein the CRISPR nuclease is Cas9, Cpf1, ahomolog thereof, a modified version thereof, a codon-optimized versionthereof, or any combination thereof.

In another method, Method 58, the present disclosure provides a methodas provided in Method 56 or 57, wherein the CRISPR nuclease is a S.pyogenes Cas9.

In another method, Method 59, the present disclosure provides a methodas provided in any one of Methods 56 to 58, wherein the CRISPR nucleasecomprises an N-terminus nuclear localization signal (NLS) and/or aC-terminus NLS.

In another method, Method 60, the present disclosure provides a methodas provided in any one of Methods 56 to 59, wherein the CRISPR nucleaseand the guide RNA are present at a weight ratio of 1:1.

In another method, Method 61, the present disclosure provides a methodas provided in any one of Methods 44 or 46 to 60, wherein the deletionand/or insertion of (i) is within or near a B2M gene locus, and theinsertion of (ii) is a polynucleotide encoding PD-L1.

In another method, Method 62, the present disclosure provides a methodas provided in Method 61, wherein the guide RNA used for (i) and (ii)comprises a nucleotide sequence comprising at least one of SEQ ID NOS:1-3 or 35-44.

In another method, Method 63, the present disclosure provides a methodas provided in Method 61 or 62, wherein the polynucleotide encodingPD-L1 is flanked by (a) a nucleotide sequence having sequence homologywith a region located left of the site in (i) and (b) a nucleotidesequence having sequence homology with a region located right of thesite in (i).

In another method, Method 64, the present disclosure provides a methodas provided in Method 63, wherein the polynucleotide encoding PD-L1 isinserted into the B2M gene locus within 50 base pairs of the site in(i).

In another method, Method 65, the present disclosure provides a methodas provided in Method 63 or 64, wherein (a) consists essentially of anucleotide sequence of SEQ ID NO: 13, and (b) consists essentially of anucleotide sequence of SEQ ID NO: 19.

In another method, Method 66, the present disclosure provides a methodas provided in any one of Methods 61 to 65, wherein the polynucleotideencoding PD-L1 is operably linked to an exogenous promoter, optionallywherein the exogenous promoter is a CAG promoter.

In another method, Method 67, the present disclosure provides a methodas provided in any one of Methods 44 to 66, wherein the cell is amammalian cell, optionally wherein the cell is a human cell.

In another method, Method 68, the present disclosure provides a methodas provided in any one of Methods 44 to 67, wherein the cell is a stemcell.

In another method, Method 69, the present disclosure provides a methodas provided in any one of Methods 44 to 68, wherein the cell is apluripotent stem cell (PSC), an embryonic stem cell (ESC), an adult stemcell (ASC), an induced pluripotent stem cell (iPSC), or a hematopoieticstem and progenitor cell (HSPC).

In another method, Method 70, the present disclosure provides a methodas provided in any one of Methods 44 to 69, wherein the cell is adifferentiated cell or a somatic cell.

In another method, Method 71, the present disclosure provides a methodas provided in any one of Methods 44 to 69, wherein the universal donorcell is capable of being differentiated into lineage-restrictedprogenitor cells or fully differentiated somatic cells.

In another method, Method 72, the present disclosure provides a methodas provided in Method 71, wherein the lineage-restricted progenitorcells are pancreatic endoderm progenitors, pancreatic endocrineprogenitors, mesenchymal progenitor cells, muscle progenitor cells,blast cells, or neural progenitor cells.

In another method, Method 73, the present disclosure provides a methodas provided in Method 71, wherein the fully differentiated somatic cellsare endocrine secretory cells such as pancreatic beta cells, epithelialcells, endodermal cells, macrophages, hepatocytes, adipocytes, kidneycells, blood cells, or immune system cells.

In another composition, Composition 37, the present disclosure providesa composition comprising a plurality of universal donor cells generatedby a method as provided in any one of Methods 44 to 73.

In another composition, Composition 38, the present disclosure providesa composition as provided in Composition 37, wherein the plurality ofuniversal donor cells can be maintained for a time and under conditionssufficient for the cells to undergo differentiation.

In another composition, Composition 39, the present disclosure providesa composition comprising cells that comprise (i) at least one deletionwithin or near at least one gene that encodes one or more MHC-1 andMHC-II human leukocyte antigens or a components or a transcriptionalregulator of a MHC-I or MHC-II complex; and (ii) at least one insertionof a polynucleotide that encodes at least one tolerogenic factor at asite that partially overlaps, completely overlaps, or is containedwithin, the site of a genetic deletion of (i).

In another method, Method 74, the present disclosure provides a methodcomprising administering to a subject the plurality of universal donorcells of Composition 37 or 38.

In another method, Method 75, the present disclosure provides a methodfor treating of a subject in need thereof, the method comprising (i)obtaining or having obtained the plurality of universal donor cells ofComposition 37 or 38 following differentiation into lineage-restrictedprogenitor cells or fully differentiated somatic cells; and (ii)administering the lineage-restricted progenitor cells or fullydifferentiated somatic cells to the subject.

In another method, Method 76, the present disclosure provides a methodof obtaining cells for administration to a subject in need thereof, themethod comprising (i) obtaining or having obtained the universal donorcells of Composition 37 or 38; and (ii) maintaining the universal donorcells for a time and under conditions sufficient for the cells todifferentiate into lineage-restricted progenitor cells or fullydifferentiated somatic cells.

In another method, Method 77, the present disclosure provides a methodas provided in Method 75 or 76, wherein the lineage-restrictedprogenitor cells are pancreatic endoderm progenitors, pancreaticendocrine progenitors, mesenchymal progenitor cells, muscle progenitorcells, blast cells, or neural progenitor cells.

In another method, Method 78, the present disclosure provides a methodas provided in Method 75 or 76, wherein the fully differentiated somaticcells are endocrine secretory cells such as pancreatic beta cells,epithelial cells, endodermal cells, macrophages, hepatocytes,adipocytes, kidney cells, blood cells, or immune system cells.

In another method, Method 79, the present disclosure provides a methodas provided in any one of Methods 74 to 78, wherein the subject is ahuman who has, is suspected of having, or is at risk for a disease.

In another method, Method 80, the present disclosure provides a methodas provided in Method 79, wherein the disease is a geneticallyinheritable disease.

In another method, Method 81, the present disclosure provides a methodfor generating a universal donor cell, the method comprising deliveringto a pluripotent stem cell (PSC) (a) an RNA-guided nuclease; (b) a guideRNA (gRNA) targeting a target site in a beta-2-microglobulin (B2M) genelocus; and (c) a vector comprising a nucleic acid, the nucleic acidcomprising (i) a nucleotide sequence homologous with a region locatedleft of the target site in the B2M gene locus, (ii) a nucleotidesequence encoding a tolerogenic factor, and (iii) a nucleotide sequencehomologous with a region located right of the target site in the B2Mgene locus, wherein the B2M gene locus is cleaved at the target site andthe nucleic acid is inserted into the B2M gene locus within 50 basepairs of the target site, thereby generating a universal donor cell,wherein the universal donor cell has increased immune evasion and/orcell survival compared to a PSC that does not comprise the nucleic acidinserted into the B2M gene locus.

In another method, Method 82, the present disclosure provides a methodas provided in Method 81, wherein the gRNA comprises a nucleotidesequence chosen from SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3.

In another method, Method 83, the present disclosure provides a methodas provided in Method 81 or 82, wherein (i) consists essentially of anucleotide sequence of SEQ ID NO: 13, and (iii) consists essentially ofa nucleotide sequence of SEQ ID NO: 19.

In another method, Method 84, the present disclosure provides a methodas provided in any one of Methods 81 to 83, wherein the tolerogenicfactor is programmed death ligand 1 (PD-L1) or human leukocyte antigen E(HLA-E).

In another method, Method 85, the present disclosure provides a methodas provided in any one of Methods 81 to 84, wherein the nucleotidesequence encoding the tolerogenic factor is operably linked to anexogenous promoter.

In another method, Method 86, the present disclosure provides a methodas provided in Method 85, wherein the exogenous promoter isconstitutive, cell type-specific, tissue-type specific, or temporallyregulated.

In another method, Method 87, the present disclosure provides a methodas provided in Method 85 or 86, wherein the exogenous promoter is a CAGpromoter.

In another method, Method 88, the present disclosure provides a methodas provided in any one of Methods 81 to 87, wherein the vector is aplasmid vector.

In another method, Method 89, the present disclosure provides a methodas provided in Method 88, wherein the plasmid vector comprises anucleotide sequence of SEQ ID NO: 33 or SEQ ID NO: 34.

In another method, Method 90, the present disclosure provides a methodas provided in any one of Methods 81 to 89, wherein the RNA-guidednuclease is a Cas9 nuclease.

In another method, Method 91, the present disclosure provides a methodas provided in Method 90, wherein the Cas9 nuclease is linked to atleast one nuclear localization signal (NLS).

In another method, Method 92, the present disclosure provides a methodas provided in Method 90 or 91, wherein the Cas9 nuclease is a S.pyogenes Cas9.

In another method, Method 93, the present disclosure provides a methodas provided in any one of Methods 81 to 92, wherein the PSC is anembryonic stem cell (ESC), an adult stem cell (ASC), an inducedpluripotent stem cell (iPSC), or a hematopoietic stem and progenitorcell (HSPC).

In another method, Method 94, the present disclosure provides a methodas provided in any one of Methods 81 to 93, wherein the PSC is a humanPSC.

In another method, Method 95, the present disclosure provides a methodfor generating a universal donor cell, the method comprising deliveringto a pluripotent stem cell (PSC) (a) an RNA-guided nuclease; (b) a guideRNA (gRNA) targeting a target site in a beta-2-microglobulin (B2M) genelocus, wherein the gRNA comprises a nucleotide sequence of SEQ ID NO: 2;and (c) a vector comprising a nucleic acid, the nucleic acid comprising(i) a nucleotide sequence homologous with a region located left of thetarget site in the B2M gene locus that consists essentially of SEQ IDNO: 13, (ii) a nucleotide sequence encoding a tolerogenic factor, and(iii) a nucleotide sequence homologous with a region located right ofthe target site in the B2M gene locus that consists essentially of SEQID NO:19, wherein the B2M gene locus is cleaved at the target site andthe nucleic acid is inserted into the B2M gene locus within 50 basepairs of the target site, thereby generating the universal donor cell,wherein the universal donor cell has increased immune evasion and/orcell survival compared to a PSC that does not comprise the nucleic acidinserted into the B2M gene locus.

In another method, Method 96, the present disclosure provides a methodas provided in Method 95, wherein the tolerogenic factor is programmeddeath ligand 1 (PD-L1) or human leukocyte antigen E (HLA-E).

In another method, Method 97, the present disclosure provides a methodas provided in Method 95 or 96, wherein the nucleotide sequence encodingthe tolerogenic factor is operably linked to an exogenous promoter.

In another method, Method 98, the present disclosure provides a methodas provided in Method 97, wherein the exogenous promoter isconstitutive, cell type-specific, tissue-type specific, or temporallyregulated.

In another method, Method 99, the present disclosure provides a methodas provided in Method 97 or 98, wherein the exogenous promoter is a CAGpromoter.

In another method, Method 100, the present disclosure provides a methodas provided in any one of Methods 95 to 99, wherein the vector is aplasmid vector.

In another method, Method 101, the present disclosure provides a methodas provided in Method 100, wherein the plasmid vector comprises anucleotide sequence of SEQ ID NO: 33 or SEQ ID NO: 34.

In another method, Method 102, the present disclosure provides a methodas provided in any one of Methods 95 to 101, wherein the RNA-guidednuclease is a Cas9 nuclease.

In another method, Method 103, the present disclosure provides a methodas provided in Method 102, wherein the Cas9 nuclease is linked to atleast one nuclear localization signal (NLS).

In another method, Method 104, the present disclosure provides a methodas provided in Method 102 or 103, wherein the Cas9 nuclease is a S.pyogenes Cas9.

In another method, Method 105, the present disclosure provides a methodas provided in any one of Methods 95 to 104, wherein the PSC is anembryonic stem cell (ESC), an adult stem cell (ASC), an inducedpluripotent stem cell (iPSC), or a hematopoietic stem and progenitorcell (HSPC).

In another method, Method 106, the present disclosure provides a methodas provided in any one of Methods 95 to 105, wherein the PSC is a humanPSC.

VII. Examples

The examples below describe generation and characterization of universaldonor cells according to the present disclosure. Table 1 liststolerogenic factors and Table 2 lists survival factors that may begenetically modified in said cells. FIGS. 1A-1C depict various geneediting strategies that can be employed for immune evasion.

TABLE 1 Tolerogenic factors that may be genetically modified FactorKnock out (KO) Knock in (KI) HLA-E + HLA-G + CTLA-4 + CD47 + PD-L1 + B2M− HLA-ABC − CIITA −

TABLE 2 Survival factors that may be genetically modified Factor Knockout (KO) Knock in (KI) ZNF143 − TXNIP − FOXO − JNK − MANF +

Example 1: Generation of B2M Knock-Out IPSCs

Guide RNA (gRNA) Selection for B2M.

To identify a large spectrum of gRNAs capable of editing the B2M DNAtarget region, an in vitro transcribed (IVT) gRNA screen was conducted.B2M targeting gRNAs were designed to target exon 1 of the B2M gene. B2Mgenomic sequence was submitted for analysis using gRNA design software.The resulting list of gRNAs was narrowed to a list of about 200 gRNAsbased on uniqueness of sequence (only gRNAs without a perfect matchsomewhere else in the genome were screened) and minimal predicted offtargets. This set of gRNAs was in vitro transcribed and transfectedusing MessengerMax into HEK293T cells that constitutively express Cas9.Cells were harvested 48 hours post transfection, the genomic DNA wasisolated, and cutting efficiency was evaluated using TIDE analysis.Guide RNAs with high indels and low predicted off target effects wereselected for further analysis. Table 3 presents the target sequences ofselected B2M gRNAs.

TABLE 3 Selected B2M gRNA Target Sequences Name Target Sequence (5′-3′)SEQ ID NO: PAM B2M-1 gRNA GCTACTCTCTCTTTCTGGCC  1 TGG (Exon 1_T12)B2M-2 gRNA GGCCGAGATGTCTCGCTCCG  2 TGG (Exon 1_T2) B2M-3 gRNACGCGAGCACAGCTAAGGCCA  3 CGG (Exon 1_T8) Exon 1_T1 TATAAGTGGAGGCGTCGCGC35 TGG Exon 1_T3 GAGTAGCGCGAGCACAGCTA 36 AGG Exon 1_T4ACTGGACGCGTCGCGCTGGC 37 GGG Exon 1_T5 AAGTGGAGGCGTCGCGCTGG 38 CGGExon 1_T6 GGCCACGGAGCGAGACATCT 39 CGG Exon 1_T7 GCCCGAATGCTGTCAGCTTC 40AGG Exon 1_T9 CTCGCGCTACTCTCTCTTTC 41 TGG Exon 1_T10TCCTGAAGCTGACAGCATTC 42 GGG Exon 1_T11 TTCCTGAAGCTGACAGCATT 43 CGGExon 1_T13 ACTCTCTCTTTCTGGCCTGG 44 AGG

Screening of B2M gRNAs in IPSCs.

Three gRNAs (B2M-1, B2M-2, and B2M-3) were used to edit iPSCs. Thelocation of the target sequence of each of these gRNAs is diagrammed inFIG. 2. IPSCs (TC-1133 cell line, RUDCR, NJ) cells were nucleofectedusing the Lonza 4D nucleofector and the P3 primary cell kit (Lonza, cat# V4XP-3024) with an RNP mixture of Cas9 (Aldevron, cat #9212-5MG) andgRNA (Synthego) at a molar ratio of 3:1 (gRNA:Cas9) with finalconcentrations of 125 pmol Cas9 and 375 pmol gRNA. Cells weredissociated using Accutase (Stempro, cat # A1110501), then resuspendedin DMEM/F12 media (Gibco, cat #11320033), counted using a Cellometer(Nexcellon) and centrifuged. Cells were resuspended in P3 buffer withsupplement 1 (4.5:1 ratio) at a concentration of 2×10³ cells/μL. A totalof 2×10⁵ cells were combined with RNP complex, transferred to anucleofection cuvette (Lonza kit) and nucleofected using program CA-137.For each cuvette 250 μL of StemFlex media (Gibco, cat # A3349401) withCloneR (Stem Cell Technologies, cat #05888) (1:10 ratio) was used toresuspend nucleofected cells. This cell suspension was split into twowells of a Vitronectin (Gibco, cat # A14700) coated 24-well plate withan additional 250 μL of StemFlex with CloneR. Cells were recovered in ahypoxic incubator (37° C., 4% O₂, 8% CO₂) for 48 hours. After 48 hours,genomic DNA was harvested from one well of each technical replicateusing a gDNA isolation kit (Qiagen, cat #69506)

The isolated gDNA was subjected to PCR to determine indel frequency. PCRfor relevant regions was performed using Platinum Taq Supermix(Invitrogen, cat #125320176 and Cat #11495017) with B2M primers. Theprimer sequences are provided in Table 4 and the locations of B2Mprimers relative to the gRNA target sites are shown in FIG. 2. Thecycling conditions are provided in Table 5.

TABLE 4 B2M TIDE Primers Name Type Sequence (5′-3′) SEQ ID NO: B2MF2forward CAGACAGCAAACTCACCCAG 4 B2MR2 reverse AAACTTTGTCCCGACCCTCC 5

TABLE 5 B2M PCR Cycling Parameters Step Temperature Time CyclesDenaturation 94° C. 2 min 1 Denaturation 94° C. 15 sec 38 Annealing 55°C. ° C. 30 sec Extension 68° C. 45 sec Elongation 68° C. 5 min 1 Hold 4hold

The resulting amplicons were submitted for PCR cleanup and Sangersequencing. Sanger sequencing results were input into Tsunami along withguide sequence. Indel percentages and identities were calculated by thesoftware (FIG. 3A). B2M-1, B2M-2, and B2M-3 gRNAs had indel frequenciesof 2.5%±1.1%, 87.6%±14.1%, and 63.9%±0.9%, respectively (n=2). FIGS. 3Band 3C presents distributions of indel outcomes for the B2M-2 (FIG. 3B)and B2M-3 gRNAs (FIG. 3C).

The cells in the duplicate well were maintained until confluent and thenpassaged to sequentially larger vessels. The bulk population wastransitioned to Advanced 20/10/10 media (See Table 6) and Laminin-521(Stem Cell Technologies, cat #77004) for maintenance.

TABLE 6 Advanced 20/10/10 Media Formulation Working Reagent ReagentAmount concentration information DMEM/F12 No HEPES 955 mL Base Gibco(11330032) Normocin 2 mL Invivogen (ANTNR1) Non-Essential Amino 10 mL1x   Gibco Acids, 100x (11140076) Chemically Defined 2 mL 0.2x GibcoLipids, 100x (11905031) 20% HSA (FAF) 5 mL 0.1%   Sigma (A1887) 7.5%Sodium bicarbonate 7 mL 0.0525% Gibco (25080094) Human insulin, 4 mg/mL5 mL 20 μg/μL Invitrogen (12585014) Sodium Chloride, 250 mg/mL 2 mL 0.5mg/mL Sigma Ascorbic Acid, 200 mM 1 mL 200 μM Sigma Holo-Transferrin, 10mg/mL 1 mL 10 μg/mL Sigma Sodium selenite, 140 μg/mL 100 μL 14 ng/mLSigma To make complete Advanced 20/10/10 (1 L volume) add the followingat time of first use FGF basic, 100 μg/mL 200 μL 20 ng/mL PeprotechActivin A, 100 μg/ML 100 μL 10 ng/mL Peprotech Heregulin, 100 μg/mL 100μL 10 ng/mL Peprotech

B2M KO IPS Clone Generation and Characterization.

Sequence-verified bulk-edited populations (FIG. 4A) were single cellsorted using a FACS-ARIA (BD Bioscience) into Vitronectin coated 96-wellplates and recovered in StemFlex with CloneR. Briefly, cells weredissociated from maintenance flasks using Accutase and resuspended inStemFlex with CloneR. Cells were then counted using Cellometer anddiluted to 1×10⁵/mL. 2 mL of this was filtered through a cell strainer(Falcon, #352235) into a FACS tube, provided to the operator and singlecells were sorted into individual wells. Plated single cells were grownin a hypoxic incubator (37° C., 8% CO₂, 4% O₂) with every other daymedia changes until colonies were large enough to be re-seeded as singlecells. When confluent, samples were split for maintenance and gDNAextraction (see above). Clone identity was confirmed via PCR and Sangersequencing (see below for details). Table 7 presents the sequences ofselect clones around the cut site (sequences deleted and/or inserted areshown in bold).

TABLE 7 Sequence Analysis of B2M KO Clones SEQ ID Clone Sequence (5′-3′)NO: WT CT XCGC        T CCGTGGZGCTA, X = N₃₀, 27 Z = N₁₆ A9CT XCGCGCTACTTA- -----GZGCTA, X = N₃₀, 28 Z = N₁₆ A11CT XCGC        TTCCGTGGZGCTA, X = N₃₀, 29 Z = N₁₆ B10CTAA----       - ----GGZGCTA, X = N₃₀, 30 Z = N₁₆ B12CT XCGC        TTCCGTGGZGCTA, X = N₃₀, 29 Z = N₁₆ C5CT XCGC        - -------GCTA, X = N₃₀, 31 Z = N₁₆ C9CT XCGC        - CCGTGGZGCTA, X = N₃₀, 32 Z = N₁₆ C11CT XCGC        TTCCGTGGZGCTA, X = N₃₀, 29 Z = N₁₆ C12CT XCGC        TTCCGTGGZGCTA, X = N₃₀, 29 Z = N₁₆

Clone sequences were aligned in Snapgene software to determine indelidentity and homo- or heterozygosity. As shown in FIG. 4B, 8 clones werehomozygous for B2M KO and 7 clones were heterozygous for B2M KO.Homozygous clones with desired edits were expanded and further verifiedthrough sequencing and flow cytometry. Clones were initially maintainedin StemFlex media on Vitronectin coated plates then eventuallytransitioned to Advanced 20/10/10 medium and Laminin-521 coated vessels.

Cells were further maintained on Laminin-521 coated flasks with Advanced20/10/10. Edited clones were verified for indel identity through PCR ofthe B2M region and Sanger sequencing. Knockout was verified through FlowCytometry for B2M and HLA-A (See Tables 8 and 9 for list of antibodiesutilized) and Taqman qPCR analysis of B2M expression using standardTaqman protocols (Taqman FastAdvanced Mastermix, ThermoFisher, cat#4444556). Levels of B2M expression for three B2M KO clones as well aswild type (unmodified) cells is presented in FIG. 5. All three KO clonestested showed decreased mRNA expression of B2M relative to wild typecell.

TABLE 8 Antibodies for Pluripotency Flow Cytometry Target FluorophoreManufacturer Catalog number SSEA-4 AlexaFluor 647 ThermoFisher SSEA421Tra-1-60 PE ThermoFisher MA1-023-PE Tra-1-81 PE BD Bioscience 560161

TABLE 9 Antibodies for B2M and HLA-ABC Target Fluorophore ManufacturerCatalog number B2M AlexaFluor 647 Biolegend 316311 HLA-ABC FITC BDPharmigen 555552

RNA extraction was carried out using Qiagen RNeasy kit with RNase-FreeDNase according to manufacturer's instructions (Qiagen, cat #74104 and79254). cDNA synthesis was carried out using Advanced iScript cDNAsynthesis kit for RT-qPCR (BioRad, cat #1725037) according tomanufacturer's instructions. Karyotypic status of clones was evaluatedthrough Karyostat service (ThermoFisher) and tracking of knownkaryotypic abnormality BCL2L1 through ddPCR using manufacturer'sinstructions and ddPCR supermix for Probes (no dUTP) (BioRad, cat#1863024; Primers in Table 10) with an annealing temperature of 59° C.and RPP30 as a reference assay.

TABLE 10 ddPCR Primer Probe Sets BCL2L1 (Target) SEQ ID NO:RPP30 (Reference) SEQ ID NO: Forward TCTGCAGAAGGCTACCC 7GATTTGGACCTGCGAGCG  9 Primer CTA Reverse TGCTGTGTCTAAGACCTC 8CAAGCCTGGCAATAAACA 10 Primer TTTCAT ATGA Probe Universal probe — 5′ VIC-11 #44 (Sigma, CTGCTGCCTGAACAT-3′- cat# 4688040001) MGB-NFQ

Resulting amplicons were gel-checked on a pre-cast 2% agarose gel(ThermoFisher, cat # G501802) and submitted for PCR cleanup and Sangersequencing. Resulting sequencing files were input into Tsunami softwarealong with gRNA sequence and a control sequence file to determine indelidentity and percentage.

Clones were also confirmed to be negative for expression of B2M and MHCClass I antigens (HLA-A, B, C), with or without Interferon-gammatreatment (25 ng/mL, R & D Systems, 285-IF) through flow cytometry ofthe same. See FIGS. 6A-6D.

Clones were confirmed to retain pluripotency through flow cytometry forpluripotency cell surface markers (FIGS. 7A-7D). Additional confirmationof pluripotency included Taqman Scorecard (ThermoFisher, cat # A15872),Thermo Pluritest service and Trilineage differentiation (see below forfull protocol).

Cells were dissociated and counted as above, prior to centrifugation andresuspension in Advanced 20/10/10 media with 2 μM Y-27632 (Tocris, cat#1245) up to 1×10⁶/mL. Resuspended cells were then filtered through a 40μM filter (Fisherbrand, cat #22363547) and 5 mL of the suspension wasplated in a single well of an ultra-low attachment 6-well dish (Corning,cat #3471). The cells were then placed on an orbital shaker overnight at98 RPM to form aggregates. After 16 hours, spent media was removed fromeach well by carefully swirling plate to collect aggregates. 4 mL offresh Advanced 20/10/10 was added.

After 24 additional hours, the cells were differentiated. Aggregateswere first collected into a 50 mL conical tube and centrifuged at 1000RPM for 1 min to settle aggregates. Media was aspirated and aggregateswere washed with DMEM/F12. Aggregates were again collected bycentrifugation and resuspended in 4 mL of respective differentiationmedia before being returned to dish and shaker. All differentiationsused the following base media: 480 mL IMDM+Glutamax (Gibco, cat#31980030), 480 mL F12+Glutamax (Gibco, cat #31765035), 10 mL ofNon-essential amino acids (Gibco, cat #11140076), 5 mL of 20% BSA(Sigma, cat # A7638-5G), 2 mL Chemically defined Lipids (Gibco, cat#11905031), 1 mL of 200 mM Ascorbic Acid (Sigma, cat # A4403-100MG), 1mL of 10 mg/mL holo-transferrin (Sigma, cat # T0665), and 100 μL of 140μg/ml sodium selenite (Sigma, cat #55261). To differentiate cells intoan ectoderm cell, a final concentration of 4 mg/mL insulin (Gibco, cat#12585014), 2 μM A83-01, 2 μM Dorsomorphin (Peprotech, cat #8666430) and2 μM PNU-74654 were used for two days. To differentiate cells into amesoderm cell, a final concentration of 1 μg/mL Insulin, 0.1 μM PIK-90,3 μM CHIR99021 (Peprotech, cat #2520691) and 0.5 μM LDN193189(Peprotech, cat #1062443) was used for two days. For day 1 of endodermdifferentiation, a final concentration of 0.2 μg/mL insulin, 0.1 μMPIK-90, 100 ng/mL Activin-A (Peprotech, cat #120-00), 2 μM CHIR99021 and20 ng/mL of FGF basic (Peprotech, cat #101-18b) was used. An additionaltwo days of differentiation for endoderm were carried out with: 0.2μg/mL insulin, 0.1 μM PIK-90, 100 ng/mL Activin A and 0.25 μM LDN193189.Media was changed daily for all differentiations. All were collected forRNA analysis using Taqman Scorecard at day 3.

Example 2: Cell Maintenance and Expansion

Maintenance of hESC/hiPSCs.

Cells of human embryonic stem cell (hESCs) line CyT49 were maintained,cultured, passaged, proliferated, and plated as described in Schulz etal. (2012) PLoS ONE 7(5): e37004. CyT49 cells were disassociated usingACCUTASE® (Stemcell Technologies 07920 or equivalent).

Human induced pluripotent stem cells (hiPSCs), such as the TC1133 cellline (Lonza), were maintained in StemFlex Complete (Life Technologies,A3349401) on BIOLAMININ 521 CTG (BioLamina Cat # CT521) coated tissueculture plates. The plates were pre-coated with a 1:10 or a 1:20dilution of BIOLAMININ in DPBS, calcium, magnesium (Life Technologies,14040133) for 2 hours at 37° C. The cells were fed daily with StemFlexmedia. For passaging of the cells, same densities of cells as for CyT49were used. For plating of the cells as single cells, the cells wereplated with 1% RevitaCell™ Supplement (100×) (Thermofisher Cat #A2644501) in StemFlex on BIOLAMININ coated plates.

Single Cell Cloning of hPSCs.

For single cell cloning, hPSCs (hESCs or hiPSCs) were fed with StemFlexComplete with Revitacell (for a final concentration of 1× Revitacell)3-4 hours prior to dissociation with ACCUTASE®. Following dissociation,the cells were sorted as a single cell per well of a BIOLAMININ coated96 well tissue culture plate. The WOLF FACS-sorter (Nanocellect) wasused to sort single cells into the wells. The plates were pre-filledwith 100-200 μL of StemFlex Complete with Revitacell. Three days postcell seeding, the cells were fed with fresh StemFlex and continued to befed every other day with 100-200 μL of media. After 10 days of growth,the cells were fed daily with StemFlex until day 12-14. At this time,the plates were dissociated with ACCUTASE® and the collected cellsuspensions were split 1:2 with half going into a new 96 well plate formaintenance and half going into a DNA extraction solution QuickExtract™DNA Extraction Solution (Lucigen). Following DNA extraction, PCR wasperformed to assess presence or absence of desired gene edits at thetargeted DNA locus. Sanger sequencing was used to verify desired edits.

Expansion of Single Cell Derived hPSCs Clones.

For CyT49, successfully targeted clones were passaged onto 24-wellplates with pure 10% XF KSR A10H10 media but on BIOLAMININ-coatedplates. Following the 24-well stage, CyT49 clones were passaged asdescribed in Schulz et al. (2012) PLoS ONE 7(5): e37004.

For hiPSCs (TC1133), cells were maintained in StemFlex Completethroughout the cloning and regular maintenance processes onBIOLAMININ-coated plates with Revitacell at the passaging stages.

Example 3: Generation of B2M Knock-Out Human Pluripotent Stem Cells(hPSCs)

Guide RNA (gRNA) Selection for B2M in hPSCs.

The three B2M targeting gRNAs described above in Example 1 were used totarget the B2M gene in hPSCs. To assess their cutting efficiency inhPSCs, CyT49 cells were electroporated using the Neon Electroporator(Neon Transfection Kit ThermoFisher Cat # MPK5000) with aribonucleoprotein (RNP) mixture of Cas9 protein (Biomay) and guide RNA(Synthego) at a molar ratio of 3:1 (gRNA:Cas9) with absolute values of125 pmol Cas9 and 375 pmol gRNA. To form the RNP complex, gRNA and Cas9were combined in one vessel with R-buffer (Neon Transfection Kit) to atotal volume of 25 and incubated for 15 min at RT. Cells weredissociated using ACCUTASE®, then resuspended in DMEM/F12 media (Gibco,cat #11320033), counted using an NC-200 (Chemometec) and centrifuged. Atotal of 1×10⁶ cells were resuspended with the RNP complex and R-bufferwas added to a total volume of 125 μL. This mixture was thenelectroporated with 2 pulses for 30 ms at 1100 V. Followingelectroporation, the cells were pipetted out into an Eppendorf tubefilled with StemFlex media with RevitaCell. This cell suspension wasthen plated into tissue culture dishes pre-coated with BIOLAMININ 521CTG at 1:20 dilution. Cells were cultured in a normoxia incubator (37°C., 8% CO₂) for 48 hours. After 48 hours, genomic DNA was harvested fromthe cells using QuickExtract (Lucigen, Middleton, Wis.; Cat # QE09050).

PCR for the target B2M sequence was performed and the resultingamplified DNA was assessed for cutting efficiency by TIDE analysis. PCRfor relevant regions was performed using Platinum Taq Supermix(Invitrogen, cat #125320176 and Cat #11495017). The sequences of the PCRprimers are presented in Table 4; and the cycling conditions provided inTable 5. The resulting amplicons were submitted for PCR cleanup andSanger sequencing. Sanger sequencing results were input into Tsunamisoftware along with the guide sequence. Indel percentages and identitieswere calculated by the software. Particular gRNAs were then selectedbased on their indel frequency in hPSCs. FIG. 8 shows the cuttingefficiency for the 3 B2M gRNAs.

Off-targets of the selected gRNAs were assessed in the stem cell-derivedDNA using hybrid capture analysis of the sequence similarity predictedsites. B2M-2 and B2M-3 guides did not show detectable off-targeteffects. B2M-2 gRNA was chosen for further clone generation due to itshigh on-target activity and undetectable off-target activity.

B2M KO hPSC Clone Generation and Characterization.

Using B2M-2 gRNA, CyT49 hESCs were electroporated and single-cell sorted3 days post electroporation using the WOLF FACS-sorter (Nanocellect)into BIOLAMININ 521 CTG coated 96-well plates with StemFlex andRevitacell. Plated single cells were grown in a normoxia incubator (37°C., 8% CO₂) with every other day media changes until colonies were largeenough to be re-seeded as single cells. When confluent, samples weresplit for maintenance and genomic DNA extraction.

The B2M KO state of clones was confirmed via PCR and Sanger sequencing.The resulting DNA sequences of the target B2M region were aligned inSnapgene software to determine indel identity and homo- orheterozygosity. Clones with desired edits were expanded and furtherverified through flow cytometry assessment for B2M expression (See Table11 for list of antibodies utilized). Clones were assessed with orwithout Interferon-gamma treatment (25 ng/mL, R & D Systems, 285-IF).FIG. 9A shows B2M expression in wild type cells and FIG. 9B presents B2Mexpression in KO cells. Karyotypic status of clones was evaluatedthrough Cell Line Genetics service (Madison, Wis.) and normal karyotypewas reported.

TABLE 11 Antibodies for Pluripotency Flow Cytometry Antigen CloneFluorophore Manufacturer Catalog # Oct3/4 40/3 Alexa 647 BD Bioscience560329 SOX2 030-678 PE BD Bioscience 562195 B2M 2M2 PE Biolegend 316305HLA-ABC W6/32 Alexa 488 Biolegend 311415 mIgG1 kappa N/A PE BDBioscience 555749 PD-L1 B7-H1 Alexa-488 ThermoFisher 53-5983-42 HLA-E3D12 PE ThermoFisher 12-9953-42

Clones were confirmed to retain pluripotency through intracellular flowcytometry for pluripotency markers OCT4 and SOX2. Confirmed pluripotentclones were differentiated to pancreatic endocrine progenitors usingpreviously established methods (Schulz et al. (2012) PLoS ONE 7(5):e37004).

Example 4: Generation of B2M Knock-Out PD-L1 Knock-in Human PluripotentStem Cells (hPSCs)

Design of B2M-KO PD-L1-KI Strategy.

Plasmid design to insert PD-L1 (CD274) into the B2M locus was made suchthat the starting codon of B2M was removed after undergoing homologydirected repair (HDR) to insert PD-L1, nullifying any chance of partialB2M expression. FIG. 10 presents a schematic of the plasmid and Table 12identifies the elements and locations therein. The donor plasmidcontained a CAGGS promoter driven cDNA of PD-L1 flanked by 800 base pairhomology arms with identical sequence to the B2M locus around exon 1.The complete sequence of the plasmid is presented in SEQ ID NO: 33.

TABLE 12 Elements of B2M-CAGGS-PD-L1 Donor Plasmid Element Location(size in bp) SEQ ID NO: Left ITR 1-130 (130) 12 LHA-B2M 145-944 (800) 13CMV enhancer 973-1352 (380) 14 chicken β-actin promoter 1355-1630 (276)15 chimeric intron 1631-2639 (1009) 16 PD-L1 2684-3556 (873) 17 bGHpoly(A) signal 3574-3798 (225) 18 RHA-B2M 3805-4604 (800) 19 Right ITR4646-4786 (141) 20

The B2M-2 gRNA was used to facilitate insertion of the PD-L1 transgeneat the targeted B2M locus. The PD-L1 donor plasmid was introduced alongwith the RNP complex made up of the B2M targeting gRNA and Cas9 protein.Per 1 million of CyT49 cells, 4 μg of plasmid DNA was delivered alongwith the RNP. Electroporation was carried out as described in Example 3.Seven days post electroporation, the cells were sorted for PD-L1 surfaceexpression using the WOLF FACS-sorter (Nanocellect) into BIOLAMININ 521CTG coated 96-well plates with StemFlex with Revitacell. ForFACS-sorting, unedited cells served as a negative control. PD-L1positive cells were selected for sorting and single cell cloning.

To detect the PD-L1 surface expression, anti-PD-L1 fluorescentantibodies were used (see Table 11). Plated single cells were grown in anormoxia incubator (37° C., 8% CO₂) with every other day media changesuntil colonies were large enough to be re-seeded as single cells. Whenconfluent, samples were split for maintenance and genomic DNAextraction.

Correctly targeted clones were identified via PCR for the PD-L1 knock-in(KI) insertion using primers that amplify a region from outside theplasmid homology arms to the PD-L1 cDNA insertion enabling amplificationof the KI integrated DNA only. On-target insertion was tested forzygosity by PCR to assess if KI occurred in a heterozygous or homozygousmanner. If a heterozygous clone was identified, the KI negative allelewas sent for Sanger sequencing to verify that it contained aB2M-disrupting indel. The correct KI clones with full B2M disruption(either via KI insertion or indel formation) were expanded in increasingtissue culture formats until a population size of 30 million cells wasreached. Approximately 10 clones were expanded in this manner andconfirmed to be pluripotent by testing for OCT4 and SOX2 viaintracellular flow cytometry (FIG. 11). Clones that passed the abovetests, were then tested further for karyotypic analysis (Cell LineGenetics), as described below. Additionally, the clones were then testedfor their competence to differentiate to pancreatic endoderm precursors(PEC) via the established protocol (Schulz et al. (2012) PLoS ONE 7(5):e37004), as described below. The loss of B2M was further confirmed bylack of expression of B2M with or without interferon-gamma treatment (25ng/mL, R & D Systems, 285-IF) through flow cytometry. FIGS. 12A and 12Bshow PD-L1 expression in wildtype and B2M KO/PD-L1 KI cells,respectively.

Example 5: Generation of B2M Knock-Out HLA-E Knock-in Human PluripotentStem Cells (hPSCs)

Design of B2M-KO HLA-E-KI Strategy.

Plasmid design to insert HLA-E trimer into the B2M locus was made suchthat the starting codon of B2M was removed after undergoing homologydirected repair (HDR) to insert the HLA-E trimer, nullifying any chanceof partial B2M expression. FIG. 13 presents a schematic of the plasmidand Table 13 identifies the elements and locations therein. The HLA-Etrimer cDNA was composed of a B2M signal peptide fused to an HLA-Gpresentation peptide fused to the B2M membrane protein fused to theHLA-E protein without its signal peptide. This trimer design has beenpreviously published (Gornalusse et al. (2017) Nat. Biotechnol. 35(8):765-772). The donor plasmid for HLA-E delivery contains a CAGGS promoterdriving expression of the HLA-E trimer flanked by 800 base pair homologyarms with identical sequence to the B2M locus around exon 1. Thecomplete sequence of the plasmid is presented in SEQ ID NO: 34.

TABLE 13 Elements of B2M-CAGGS-HLA-E Donor Plasmid Element Location(size in bp) SEQ ID NO: Left ITR 1-130 (130) 12 LHA-B2M 145-944 (800) 13CMV enhancer 973-1352 (380) 14 chicken β-actin promoter 1355-1630 (276)15 chimeric intron 1631-2639 (1009) 16 B2M signal sequence 2684-2743(60) 21 HLA-G peptide 2744-2770 (27) 22 GS Linker 2771-2815 (45) 23 B2Mmembrane protein 2816-3112 (297) 24 GS Linker 3113-3172 (60) 25 HLA-E3173-4183 (1011) 26 bGH poly(A) signal 4204-4428 (225) 18 RHA-B2M4435-5234 (800) 19 Right ITR 5276-5416 (141) 20

The B2M-2 gRNA was used to facilitate insertion of the HLA-E transgeneat the targeted B2M locus. The HLA-E donor plasmid was introduced alongwith the RNP complex made up of the B2M targeting gRNA and Cas9 protein.Per 1 million of CyT49 cells, 4 μg of plasmid DNA was delivered alongwith the RNP. Electroporation was carried out as described in Example 3.Seven days post electroporation, the cells were sorted for HLA-E surfaceexpression using the WOLF FACS-sorter (Nanocellect) into BIOLAMININ 521CTG coated 96-well plates with StemFlex with Revitacell. ForFACS-sorting, unedited cells served as a negative control. HLA-Epositive cells were selected for sorting and single cell cloning.

To detect the HLA-E surface expression, anti-HLA-E fluorescentantibodies were used (Table 11). Plated single cells were grown in anormoxia incubator (37° C., 8% CO₂) with every other day media changesuntil colonies were large enough to be re-seeded as single cells. Whenconfluent, samples were split for maintenance and genomic DNAextraction.

Correctly targeted clones were identified via PCR for the HLA-E knock-in(KI) insertion using primers that amplify a region from outside theplasmid homology arms to the HLA-E cDNA insertion enabling amplificationof the KI integrated DNA only. On-target insertion was tested forzygosity by PCR to assess if KI occurred in a heterozygous or homozygousmanner. If a heterozygous clone was identified, the KI negative allelewas sent for Sanger sequencing to verify that it contained aB2M-disrupting indel. The correct KI clones with full B2M disruption(either via KI insertion or indel formation) were expanded in increasingtissue culture formats until a population size of 30 million cells wasreached. Approximately 10 clones were expanded in this manner andconfirmed to be pluripotent by testing for OCT4 and SOX2 viaintracellular flow cytometry (FIG. 14). Clones that passed the abovetests were then tested further for karyotypic analysis (Cell LineGenetics). Additionally, the clones were tested for their competence todifferentiate to pancreatic endoderm precursors (PEC) via theestablished protocol (Schulz et al. (2012) PLoS ONE 7(5): e37004). Theloss of B2M was further confirmed by lack of expression of HLA-A,B,Cwith or without interferon-gamma treatment (50 ng/mL, R & D Systems,285-IF) through flow cytometry (FIG. 15). FIG. 16 shows HLA-Eexpression.

Example 6: Karyotype Analysis of Edited Clones

G-Band Karyotyping Analysis of Edited Embryonic Stem (ES) Cells.

1 million of edited ES cells were passaged into a T-25 culture flaskwith culture media (DMEM/F12+10% Xeno-free KSR with 10 ng/mL Activin and10 ng/mL Heregulin). After culturing overnight, three T25 culture flaskswere shipped to Cytogenetics Laboratory (Cell Line Genetics, Inc.) forKaryotyping analysis; FISH analysis for Chromosome 1, 12, 17, 20; andarray comparative genomic hybridization (aCGH) analysis with standard8×60K array. The G-banding results of selected cells electroporated withnon-cutting guides (“NCG”), B2M KO clones, B2M HO/PD-L1 HI clones(“V1-A”), and B2M KO/HLA-E KI clones (“V2-A”) are shown in Table 14.

TABLE 14 G-band Karyotyping Results Karyo- aCGH typing FISH array CellLine Type Passage analysis analysis analysis NCG#1 non-cutting guide P36Normal Normal PASS NCG#2 non-cutting guide P36 Normal Normal PASSB2MKO#1 B2M KO P38 Normal Normal PASS B2MKO#2 B2M KO P36 Normal NormalPASS B2MKO#3 B2M KO P36 Normal Normal PASS V1-A003 B2M KO/PD-L1 KI P37Normal Normal PASS V1-A004 B2M KO/PD-L1 KI P38 Normal Normal PASSV1-A007 B2M KO/PD-L1 KI P37 Normal Normal PASS V1-A008 B2M KO/PD-L1 KIP38 Normal Normal PASS V2-A005 B2M KO/HLA-E KI P42 Normal Normal PASSV2-A006 B2M KO/HLA-E KI P38 Normal Normal PASS V2-A007 B2M KO/HLA-E KIP38 Normal Normal PASS

Example 7: Differentiation of Edited Human Embryonic Stem Cells toPancreatic Endoderm Cells (PECs)

Maintenance of Edited Human Embryonic Stem Cells (ES).

The edited human embryonic stem cells at various passages (P38-42) wereseeded at 33,000 cells/cm² for a 4-day passage or 50,000 cells/cm² for a3-day passage with hESM medium (DMEM/F12+10% KSR+10 ng/mL Activin A and10 ng/mL Heregulin) and final 10% human AB serum.

Aggregation of Edited Human Embryonic Stem Cells for PECsDifferentiation.

The edited ES were dissociated into single cells with ACCUTASE® and thencentrifuged and resuspended in 2% StemPro (Cat # A1000701, Invitrogen,CA) in DMEM/F12 medium at 1 million cells per ml, and total 350-400million of cells were seeded in one 850 cm² roller bottle (Cat #431198,Corning, N.Y.) with rotation speed at 8RPM±0.5RPM for 18-20 hours beforedifferentiation. The ES aggregates from edited human embryonic stemcells were differentiation into pancreatic lineages using roller bottlesas described in Schulz et al. (2012) PLoS ONE 7(5): e37004.

Example 8: Characterization of Differentiated Pancreatic Endoderm Cells(PECs)

Flow Cytometry for FOXA2 and SOX17 at Stage 1 (DE) and CHGA, PDX1 andNKX6.1 at PEC Stage.

hESC-derived stage 1 aggregates, or hESC-derived pancreatic aggregates,were washed with PBS and then enzymatically dissociated to single cellssuspension at 37° C. using ACCUMAX™ (Catalog # A7089, Sigma, MO). MACSSeparation Buffer (Cat #130-091-221, Miltenyi Biotec, NorthRhine-Westphalia, Germany) was added and the suspension was passedthrough a 40 μm filter and pelleted. For intracellular marker staining,cells were fixed for 30 mins in 4% (wt/v) paraformaldehyde, washed inFACS Buffer (PBS, 0.1% (wt/v) BSA, 0.1% (wt/v) NaN₃) and then cells werepermeabilized with Perm Buffer (PBS, 0.2% (v/v) Triton X-100 (Cat #A16046, Alfa Aesar, MA), 5% (v/v) normal donkey serum, 0.1% (wt/v) NaN3)for 30 mins on ice and then washed with washing buffer (PBS, 1% (wt/v)BSA, 0.1% (wt/v) NaN₃). Cells were incubated with primary antibodies(Table 15) diluted with Block Buffer (PBS, 0.1% (v/v) Triton X-100, 5%(v/v) normal donkey serum, 0.1% (wt/v) NaN₃) overnight at 4° C. Cellswere washed in IC buffer and then incubated with appropriate secondaryantibodies for 60 mins at 4° C. Cells were washed in IC buffer and thenin FACS Buffer. Flow cytometry data were acquired with NovoCyte FlowCytometer (ACEA Biosciences, Brussels). Data were analyzed using FlowJosoftware (Tree Star, Inc.). Intact cells were identified based onforward (low angle) and side (orthogonal, 90°) light scatter. Backgroundwas estimated using antibody controls and undifferentiated cells. In thefigures, a representative flow cytometry plot is shown for one of thesub-populations. Numbers reported in the figures represent thepercentage of total cells from the intact cells gate.

TABLE 15 Antibodies for flow cytometry for characterization ofdifferentiated PECs Antigen Fluorophore Source Dilution SOX17 AF647 BDBioscience (Cat#562594) 1:50  FOXA2 PE Miltenyi Biotechnology 1:10 (Cat#130-107-773) PDX1 PE BD Bioscience (Cat#562161) 1:2.5 NKX6.1 AF647BD Bioscience (Cat#563338) 1:2.5 CHGA AF405 Novus (Cat#NBP2-33198AF405) 1:1000

At DE stage, the population of FOXA2 and SOX17 double positive cellswere more than 90% of total cells from CyT49 wild types differentiatedcells. The PD-L1 KI/B2M KO, HLA-E KI/B2M KO, and B2M KO cells showedcomparable percentage of DE compared to wild type cells (FIGS. 17A-17Band FIG. 18).

At PECs stage, flow cytometry for chromogranin (CHGA), PDX1 and NKX6.1was performed. The heterogeneous population at PEC stage includepancreatic progenitors, early endocrine (FIG. 19). From the pie chart ofheterogeneous population (FIG. 20), the distribution of cell populationsfrom differentiated edited cells (PD-L1 KI/B2M KO or B2M KO) were verysimilar to wild type cells.

Targeted RNAseq.

Targeted RNAseq for gene expression analysis was performed usingIllumina TruSeq and a custom panel of oligos targeting 111 genes. Thepanel primarily contained genes that are markers of the developmentalstages during pancreatic differentiation. At end of each differentiationstage, 10 μL APV (aggregated pellet volume) was collected and extractedusing the Qiagen RNeasy or RNeasy 96 spin column protocol, includingon-column DNase treatment. Quantification and quality control wereperformed using either the TapeStation combined with Qubit, or by usingthe Qiagen QIAxcel. 50-200 ng of RNA was processed according to theIllumina TruSeq library preparation protocol, which consists of cDNAsynthesis, hybridization of the custom oligo pool, washing, extension,ligation of the bound oligos, PCR amplification of the libraries, andclean-up of the libraries, prior to quantification and quality controlof the resulting dsDNA libraries using either the TapeStation combinedwith Qubit, or by using the Qiagen QIAxcel. The libraries weresubsequently diluted to a concentration of 4 nM and pooled, followed bydenaturing, spike in of PhiX control, and further dilution to 10-12 pMprior to loading on the Illumina MiSeq sequencer. Following thesequencing run, initial data analysis was performed automaticallythrough BaseSpace, generating raw read counts for each of the customprobes. For each gene, these read counts were then summed for all probescorresponding to that gene, with the addition of 1 read count (toprevent downstream divisions by 0). Normalization was performed to thegene SF3B2, and the reads were typically visualized as fold change vs.Stage 0. When the data was processed for principal component analysis,normalization was performed using the DEseq method. Selected geneexpression was shown in FIG. 21. The kinetic expression pattern ofFOXA2, CHGA, PDX1 and NKX6.1 from PD-L1 KI/B2M KO or B2M KO cells wassimilar to wild type cells.

Confirmation of B2M and PD-L1 Expression at PEC Stage.

At PEC stage, differentiated aggregates were stimulated with or withoutinterferon-gamma (50 ng/ml) for 48 hours. The aggregates washed with PBSand then enzymatically dissociated into single cells suspension at 37°C. using ACCUMAX™ (Catalog # A7089, Sigma, MO). MACS Separation Buffer(Cat #130-091-221, Miltenyi Biotec, North Rhine-Westphalia, Germany) wasadded and the suspension was passed through a 40 μm filter and pelleted.For surface marker staining, dissociated cells were incubated withfluorescent-conjugated antibody diluted in MACS Separation Buffer for 20mins and then washed in MACS Separation Buffer. Cells were resuspendedin FACS buffer for flow acquisition. Flow cytometry data were acquiredwith NovoCyte Flow Cytometer. As shown the FIGS. 22A-22F, B2M expressionwas below the detection limit in differentiated PECs from PD-L1 KI/B2MKO or B2M KO, and PDL1 was expressed in the differentiated PECs fromPD-L1 KI/B2M KO cells.

Immune Phenotype of PEC Cells.

At PEC stage, differentiated aggregates were stimulated with or withoutinterferon-gamma (50 ng/ml) for 48 hours. The aggregates were harvestedfor MHC class I and II staining. No MHC class II expression at PEC stagefrom wild type or edited cells (PD-L1 KI/B2M KO and B2M KO cells) (FIGS.23D-23F). The expression of HLA-ABC (MHC class I) was low (1.3% fromwild type cells) and it was highly regulated upon IFN-γ stimulation.However, HLA-ABC was not expressed even under IFN-γ stimulation in theedited cells (PD-L1 KI/B2M KO and B2M KO cells) (FIGS. 23A-23C).

Example 9: T-Cell Activation/Proliferation Assay

PEC-differentiated cells were tested for their ability to trigger animmune response via in vitro human T-cell activation/proliferationassays. Fresh donor PBMCs were purchased from Hemacare and CD3+ T-cellswere purified using the Pan T-Cell Isolation Kit, human (Miltenyi Cat#130-096-535). The isolated T-cells were labeled with CellTrace™ CFSECell Proliferation Kit Protocol (Thermofisher Cat # C34554) permanufacturer instructions and co-incubated with differentiated PEC for 5days. Dynabeads™ Human T-Activator CD3/CD28 for T-Cell Expansion andActivation (Thermofisher Cat #11161D) were used as a positive control toactivate T-cells. T-cells alone were labeled with CFSE and used as anegative control. Percent of CD3+ CFSE+ cells was measured to assesspercent of T-cell proliferation (FIGS. 24A-24D). WT PEC triggered T-cellproliferation above T-cell alone control. B2M KO and B2M KO/PD-L1 KICyT49-derived PEC did not trigger T-cell proliferation above T-cell onlycontrol showing the hypo immunogenic nature of edited cells.

The invention claimed is:
 1. An in vitro method for generating auniversal donor cell, the method comprising delivering to a stem cell:(a) an RNA-guided nuclease; (b) a guide RNA (gRNA) targeting a targetsite in a beta-2-microglobulin (B2M) gene locus; and (c) a vectorcomprising a nucleic acid, the nucleic acid comprising (i) a nucleotidesequence homologous with a region located left of the target site in theB2M gene locus, (ii) a nucleotide sequence encoding a tolerogenicfactor, and (iii) a nucleotide sequence homologous with a region locatedright of the target site in the B2M gene locus, wherein the vectorcomprises a nucleotide sequence consisting of SEQ ID NO: 33, wherein theB2M gene locus is cleaved at the target site and the nucleic acid isinserted into the B2M gene locus within 50 base pairs of the targetsite, thereby disrupting the B2M gene and generating a universal donorcell, wherein the universal donor cell has increased immune evasionand/or cell survival compared to a stem cell that does not comprise thenucleic acid inserted into the B2M gene locus.
 2. The method of claim 1,wherein the gRNA comprises a spacer sequence corresponding to a targetsequence consisting of SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO:
 3. 3.The method of claim 1, wherein the nucleotide sequence of (i) consistsessentially of SEQ ID NO: 13, and the nucleotide sequence of (iii)consists essentially of SEQ ID NO:
 19. 4. The method of claim 1, whereinthe vector is a plasmid vector.
 5. The method of claim 1, wherein theRNA-guided nuclease is a Cas9 nuclease.
 6. The method of claim 5,wherein the Cas9 nuclease is linked to at least one nuclear localizationsignal (NLS).
 7. The method of claim 6, wherein the Cas9 nuclease is aS. pyogenes Cas9.
 8. The method of claim 1, wherein the stem cell is anembryonic stem cell (ESC), an adult stem cell (ASC), an inducedpluripotent stem cell (iPSC), or a hematopoietic stem and progenitorcell (HSPC).
 9. The method of claim 1, wherein the stem cell is a humanstem cell.
 10. An in vitro method for generating a universal donor cell,the method comprising delivering to a stem cell: (a) an RNA-guidednuclease; (b) a guide RNA (gRNA) targeting a target site in abeta-2-microglobulin (B2M) gene locus, wherein the gRNA comprises aspacer sequence corresponding to a target sequence consisting of SEQ IDNO: 2; and (c) a vector comprising a nucleic acid, the nucleic acidcomprising (i) a nucleotide sequence homologous with a region locatedleft of the target site in the B2M gene locus, wherein the nucleotidesequence that consists essentially of SEQ ID NO: 13, (ii) a nucleotidesequence encoding a tolerogenic factor, and (iii) a nucleotide sequencehomologous with a region located right of the target site in the B2Mgene locus, wherein the nucleotide sequence that consists essentially ofSEQ ID NO:19, wherein the vector comprises a nucleotide sequenceconsisting of SEQ ID NO: 33, wherein the B2M gene locus is cleaved atthe target site and the nucleic acid is inserted into the B2M gene locuswithin 50 base pairs of the target site, thereby disrupting the B2M geneand generating the universal donor cell, wherein the universal donorcell has increased immune evasion and/or cell survival compared to astem cell that does not comprise the nucleic acid inserted into the B2Mgene locus.
 11. The method of claim 10, wherein the vector is a plasmidvector.
 12. The method of claim 10, wherein the RNA-guided nuclease is aCas9 nuclease.
 13. The method of claim 12, wherein the Cas9 nuclease islinked to at least one nuclear localization signal (NLS).
 14. The methodof claim 13, wherein the Cas9 nuclease is a S. pyogenes Cas9.
 15. Themethod of claim 10, wherein the stem cell is an embryonic stem cell(ESC), an adult stem cell (ASC), an induced pluripotent stem cell(iPSC), or a hematopoietic stem and progenitor cell (HSPC).
 16. Themethod of claim 10, wherein the stem cell is a human stem cell.